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Patients characteristics

The characteristics of consecutive 179 hospitalized COVID-19 patients were showed in Table 1. The comparison between serious and non-serious infection group was showed in Supplementary Table 1. Forty-three (24%) and 136 (76%) patients were diagnosed as serious and non-serious infection, respectively. The median duration of disease was 18days (range 2102) days, and duration of SARS-CoV-2 infection>18days and18days was defined as long-term (n=90) and short-term infection (n=89), respectively (Supplementary Table 2). The median time from transplantation to COVID-19 infection occurrence was 149days (range 63038) days. A total of 116 (64.8%) patients received immunosuppressants, including cyclosporin (n=85), tacrolimus (n=8), ruxolitinib (n=8), or glucocorticoids (n=32) when COVID-19 occurrence. Seventeen patients received more than 1 type of immunosuppressants. A total of 144 (80.4%) patients received anti-viral treatment (140 for Paxlovid and 4 for Azvudine). Forty serious infection cases (93.0%) received anti-viral treatment (39 for Paxlovid and 1 for Azifudine), 85 long-term infection cases (94.4%) received anti-viral treatment (83 for Paxlovid and 2 for Azifudine). Sixty-two patients received newly added drugs corticosteroid treatment, that is, 23 (53.5%) and 39 (28.7%) patients were in the serious and non-serious infection group, 9 (10.1%) and 53 (58.9%) patients were in the short- and long-term infection group, respectively.

A total of 26, 33, and 11 patients showed PGF, leukopenia and thrombocytopenia, respectively, after COVID 19 infection. The 150-day cumulative incidence of leukopenia, thrombocytopenia, and PGF was 18.4% (95% confidence interval [CI] 12.724.1%), 6.1% (95% CI 2.619.6%), and 14.5% (95% CI 9.319.7%) respectively, after COVID-19.

The 150-day cumulative incidence of PGF after COVID-19 was 11.8% (95% CI 6.417.2%) versus 23.3% (95% CI 10.536.1%) (P=0.071) between non-serious and serious infection group, which was 9.0% (95% CI 3.015.0%) versus 20.0% (95% CI 11.728.3%) (P=0.045) between short- and long-term infection group (Fig.1A).

The 150-day cumulative incidence of PGF and leukopenia after COVID-19 between short and long-term infection group. (A) PGF; (B) leukopenia. PGF poor graft function.

The 150-day cumulative incidence of leukopenia after COVID-19 was 19.9% (95% CI 13.226.6%) versus 14.0% (95% CI 3.524.5%) (P=0.325) between non-serious and serious infection group, which was 12.4% (95% CI 5.519.3%) versus 24.4% (95% CI 15.533.3%) (P=0.049) between short- and long-term infection group (Fig.1B).

The 150-day cumulative incidence of thrombocytopenia after COVID-19 was 5.1% (95% CI 1.48.8%) versus 9.3% (95% CI 0.518.1%) (P=0.331) between non-serious and serious infection group, which was 5.6% (95% CI 0.810.4%) versus 6.7% (95% CI 1.511.9%) between short- and long-term infection group (P=0.779).

The median duration of leukopenia, thrombocytopenia, and PGF was 14days (range 4118) days, 10days (range 878) days, and 17days (range 978) days, respectively. Until the last follow up, leukopenia, thrombocytopenia and PGF were still persistence 22 patients.

In multivariable analysis, after adjusted by other factors, the duration of COVID-19 was associated with PGF (hazard ratio [HR], 2.31; 95% CI 1.045.11; P=0.039) and leukopenia (HR 2.29; 95% CI 1.045.07; P=0.04) (Table 2). The other risk factors for PGF and leukopenia were showed in Supplementary Table 4.

A total of 5 patients showed aGVHD after COVID 19 infection, and the cumulative incidence of total aGVHD was 2.8% (95% CI 0.45.2%) after COVID-19. The cumulative incidence of aGVHD after COVID-19 was 3.7% (95% CI 0.56.9%) versus 0% (P=0.204), respectively, between non-serious and serious infection group, which was 3.4% (95% CI 0.47.2%) versus 2.2% (95% CI 0.95.3%) (P=0.651), respectively, between short- and long-term infection group. No risk factors were associated with aGVHD in multivariable analysis.

A total of 4, 4 and 3 patients showed mild, moderate, and severe cGVHD after COVID 19 infection, and the cumulative incidence of cGVHD was 6.70% after COVID-19. The 150-day cumulative incidence of cGVHD after COVID-19 was 7.4% (95% CI 2.911.8%) versus 4.7% (95% CI 1.711.1%), respectively, between non-serious and serious infection group (P=0.544). The 150-day cumulative incidence of cGVHD after COVID-19 was 6.7% (95% CI 1.511.9%) versus 5.6% (95% CI 0.810.4%), respectively, between short- and long-term infection group (P=0.741). No risk factors were associated with cGVHD in multivariable analysis.

A total of 34 and 7 patients showed CMV DNAemia and CMV disease (CMV pneumonia: 5, CMV gastrointestinal disease: 1, CMV encephalitis+retinitis: 1) after COVID 19 infection. The 150-day cumulative incidence of CMV DNAemia after COVID-19 was 19.9% (95% CI 13.226.6%) versus 11.6% (95% CI 1.921.3%) (P=0.204), respectively, between non-serious and serious infection group, which was 14.6% (95% CI 7.222.0%) versus 23.3% (95% CI 14.532.1%) (P=0.118), respectively, between short- and long-term infection group. The 150-day cumulative incidence of CMV disease after COVID-19 was 0.7% (95% CI 0.72.1%) versus 14.0% (95% CI 3.524.5%) (P<0.0001, Fig.2A), respectively, between non-serious and serious infection group, which was 0% versus 7.8% (95% CI 2.213.4%) (P=0.007, Fig.2B), respectively, between short- and long-term infection group. Particularly, the 150-day cumulative incidence of CMV pneumonia after COVID-19 was 0% versus 11.6% (95% CI 1.921.4%) (P<0.0001, Fig.2C), respectively, between non-serious and serious infection group, which was 0% versus 5.6% (95% CI 0.810.4%) (P=0.0245, Fig.2D), respectively, between short- and long-term infection group. In multivariable analysis, after adjusted by other factors, the severity of COVID-19 was associated with CMV disease (HR, 20.15; 95% CI 2.43167.36, P=0.005) (Table 2).

The association between COVID-19 and CMV disease. The 150-day cumulative incidence of CMV disease after COVID-19 between (A) non-serious and serious infection group; (B) short and long-term infection group. The 150-day cumulative incidence of CMV pneumonia after COVID-19 between (C) non-serious and serious infection group; (D) short and long-term infection group. CMV cytomegalovirus.

A total of 11 and 3 patients showed Epstein-Barr virus (EBV) DNAemia and EBV associated posttransplant lymphoproliferative disorders (PTLD) after COVID-19. The 150-day cumulative incidence of EBV DNAemia after COVID-19 was 7.4% (95% CI 3.011.8%) versus 0.00% (P=0.068), respectively, between non-serious and serious infection group. The 150-day cumulative incidence of EBV DNAemia after COVID-19 was 5.6% (95% CI 0.810.4%) versus 5.6% (95% CI 0.810.4%) (P=0.968), respectively, between short- and long-term infection group. All the 3 PTLD patients were in the non-serious group. No risk factors were associated with EBV DNAemia and PTLD in multivariable analysis.

A total of 27 patients died after COVID-19, and the caused were summarized in Table 3. The most common cause was infection besides of COVID-19 (n=9, 33.3%), followed by relapse (n=7, 25.9%) and COVID-19 (n=4, 14.8%).

The 150-day cumulative incidence of non-relapse mortality (NRM) after COVID-19 infection was 11.2% (95% CI 6.615.8%), which was 2.2% (95% CI 0.34.7%) and 39.5% (95% CI 24.654.4%) between non-serious and serious infection group (P<0.0001, Fig.3A), and was 2.2% (95% CI 0.95.3%) and 20.0% (95% CI 11.728.3%) (P=0.002, Fig.3B) between short- and long-term infection group.

The association between COVID-19 and survival. The 150-day cumulative incidence of NRM after COVID-19 between (A) non-serious and serious infection group; (B) short and long-term infection group. The 150-day probability of OS after COVID-19 infection between (C) non-serious and serious infection group; (D) short and long-term infection group. NRM non-relapse mortality, OS overall survival.

The 150-day probability of overall survival (OS) after COVID-19 infection was 84.9% (95% CI 79.690.2%), which was 94.9% (95% CI 91.298.6%) and 53.5% (95% CI 38.168.5%) between non-serious and serious infection group (P<0.0001, Fig.3C), and was 93.3% (95% CI 88.198.5%) and 76.7% (95% CI 67.985.5%) (P=0.002, Fig.3D) between short- and long-term infection group.

In multivariable analysis, after adjusted by other factors, the severity of COVID-19 was associated with NRM (HR, 17.26; 95% CI 4.8761.21, P<0.0001). The severity of COVID-19 were associated with OS (HR, 14.00; 95% CI 5.8733.42, P<0.0001) (Table 2), The other risk factors for NRM and OS were showed in Supplementary Table 4.

A total of 179 patients without COVID-19 infection were enrolled as controlled and the characteristics between patients with and without COVID-19 were showed in Supplementary Table 5. The 150-day probability of NRM and OS were 11.2% (95% CI 6.615.8%) versus 3.9% (95% CI 1.16.7%) with P=0.009 and 84.9% (95% CI 79.690.2%) versus 93.9% (95% CI: 88.7%99.1%) with P=0.006, respectively, for patients in the group with and without COVID-19 infection (Fig.4A,B). The probability of NRM and OS for patients without COVID-19 infections were superior to those in serious infection group or long-term infection group (Fig.4C,D).

Clinical outcomes of patients with and without COVID 19 infection. (A) The 150-day cumulative incidence of NRM in the group with and without COVID-19 infection; (B) The 150-day cumulative incidence of OS in the group with and without COVID-19 infection; (C) The 150-day cumulative incidence of NRM in the group without COVID-19 infection, serious infection and long-term infection; (D) The 150-day cumulative incidence of OS in the group without COVID-19 infection, serious infection and long-term infection, NRM non-relapse mortality, OS overall survival.

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Structural dataset and computational analysis

The experimentally determined 3D structure of the CD45 extracellular domain was retrieved from the PDB (5FMV)22. The per-residue relative solvent accessibility area was computed using a previously published algorithm45 implemented in FreeSASA46 using default parameters. Prediction of B-cell epitopes was based on BepiPred-2.0 (ref. 47) using the default threshold (0.5) for epitope residues nomination. The EV mutation sequence analysis framework48 was used to search for the CD45 sequence with the non-redundant UniProtKB database49. A multiple-sequence alignment was built using five iterations of the jackhammer HMM search algorithm50 with default significance score for the inclusion of homologous sequences.

For sgRNA cloning into the px458 host vector (a gift from F. Zhang) (Supplementary Table 1), forward and reverse primers containing complementary CRISPR RNA (crRNA) sequences flanked by BbsI restriction sites were used (Supplementary Table 4). The px458 plasmid was double digested with AgeI-HF (NEB, R3552S) and EcoRI-HF (NEB, R3101S) to eliminate the regions coding for GFP and Cas9. The px458 vector was then digested using BbsI (ThermoFisher Scientific, ER1012), gel purified and ligated with the phosphorylated and annealed crRNA oligonucleotides (called sgRNA plasmid once cloned).

To transiently overexpress CD45 mutants, we introduced each variant of interest in a plasmid expressing WT CD45RO. Briefly, we digested the pCD45RABC plasmid (Sino Biological) (Supplementary Table 1) with HindIII-HF (NEB, R3104S) and XcmI (NEB, R0533L) to remove the alternative spliced exons A, B and C. The point mutations of the human CD45 variants were then introduced into the plasmid expressing CD45RO using PCR (Supplementary Table 4).

All ligations were transformed in JM109-competent bacteria (Promega, P9751). BE plasmids were from Addgene (SPACE-NG, ABE8e-NG, ABEmax-SpRY, ABEmax-SpG, CBE4max-NG, CBE4max-SpG and xCas9(3.7)-BE4) (Supplementary Table 1).

ABE8e-NG mRNA (capped (cap 1) using CleanCap AG; fully substituted with 5-methoxy-U; 120A polyA tail) and ABE8e(TadA-8e V106W)-SpRY mRNA (capped (cap 1) using CleanCap AG 3-O-methylation; fully substituted with N1-methyl-pseudo-U; 80A polyA tail) were from Trilink Biotechnologies and Tebu-bio. We used 100-base lyophilized chemically modified sgRNAs from Synthego using their CRISPRevolution sgRNA EZ Kit service and resuspended at 100M (3.2gl1) in 1 TE buffer from Synthego (10nM Tris, 1mM EDTA, pH 8.0; chemical modifications include 2-O-methylation of the three first and last bases and 3 phosphorothioate bonds between the first three and last two bases of each sgRNA).

Cells from the BE plasmid screening were lysed in tail lysis buffer (100mM Tris pH8.5, 5mM Na-EDTA, 0.2%SDS, 200mM NaCl) containing proteinase K (Sigma-Aldrich) at 56C (1,000rpm) for 1h. The DNA was precipitated with isopropanol (1:1 volume ratio) and washed in 70% ethanol. The DNA was then resuspended in H2O and the genomic DNA concentration was measured with a NanoDrop device (Thermo Fisher).

For samples containing few cells, genomic DNA was extracted using QuickExtract (Lucigen, QE09050). Cell pellets were resuspended in 30l QuickExtract, incubated at 60C for 6min, vortexed for 1min and subsequently re-incubated at 98C for 10min.

PCR was performed using GoTaq G2 Green Master Mix (Promega, M782B). The gDNA of samples analysed by NGS was extracted using QuickExtract (Lucigen, QE09050) or the Quick-DNA 96 Plus kit (Zymo, D4070) and the genomic DNA concentration was measured with a Qubit device (Thermo Fisher).

For Sanger sequencing, different PCR primers were used depending on the CD45 exon targeted by the sgRNA and the sequencing technology (Supplementary Table 4). Sequencing of PCR amplicons was done at Microsynth and sequencing chromatograms were analysed using the EditR R package51 to quantify BE efficiencies.

For NGS, targeted amplicon libraries were generated using a three-step PCR protocol. In brief, nested PCRs were done on genomic DNA samples using KAPA HiFi HotStart polymerase (Roche) (Supplementary Table 4). After Illumina barcoding (Nextera indices, Illumina) using KAPA HiFi HotStart polymerase (Roche), PCR samples were pooled, purified using AMPure XP beads (Beckman Coulter) and quantified using Qubit dsDNA HS assay kit (Thermo Fisher). Libraries were paired-end sequenced on an Illumina Miniseq instrument using the Illumina Miniseq Mid output kit (300 cycles) with 50% PhiX spike-in (Illumina). After demultiplexing, each sample was assessed for quality using FastQC52 and processed using the CRISPResso2 tool53. For each of the samples, we provided the reference amplicon sequence (hg38) and the guide RNA sequence (reverse complement) and defined the quantification window centre to 10, the quantification window size to 15 and the plot window size to 30. We applied minimum paired end reads overlap between 10 and 200 and provided the following Trimmomatic sentence: ILLUMINACLIP:NexteraPE-PE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Finally, we used a custom R script (https://gitlab.com/JekerLab/cd45_shielding) to count and translate into amino acid each allele from the CRISPResso2 output file Alleles_frequency_table.txt in the quantification window. Alleles with less than 0.8% frequency were considered as other.

Genetic discrimination between PDX- and HSPC-derived cells was performed using the Devyser Chimerism NGS kit (Devyser) according to the manufacturers recommendations. In brief, sequencing libraries were prepared from genomic DNA targeting 24 polymorphic insertiondeletion markers distributed on 16 different chromosomes. The libraries were sequenced on a MiniSeq (Illumina) instrument using the high-throughput flow cell, generating 74-bp pair-end reads. An informative marker set was defined for the PDX donor/HSPC donor pair. It consisted of 10 markers that reliably discriminated between DNA from PDX and HSPC donor cells. The average proportion of leukaemia donor-specific reads to total reads was calculated to determine the proportion of PDX cells in each sample. It was confirmed that the genomic DNA of the mouse host did not interfere with the analysis.

Genomic DNA was extracted from human peripheral blood mononuclear cells (PBMCs) using the Puregene tissue kit (Qiagen, 158063) according to the manufacturers instructions including the proteinase-K and RNase steps (Qiagen, 158143 and 158153). CHANGE-seq-BE was adapted and modified from the original CHANGE-seq method54 to validate genome-wide activity for ABEs28. Similar to CHANGE-seq, purified genomic DNA tagmented with a custom Tn5-transposome to generate an average length of 650bp and followed by gap repair with Kapa HiFi HotStart Uracil + DNA Polymerase (KAPA Biosystems, KK2802) and Taq DNA ligase (NEB, M0208L). Gap-repaired DNA was treated with USER enzyme (NEB, M5505L) and T4 polynucleotide kinase (NEB, M0201L). Intramolecular circularization of the DNA was performed with T4 DNA ligase (NEB, M0202L) and residual linear DNA was degraded by a cocktail of exonucleases containing plasmid-safe ATP-dependent DNase (Lucigen, E3110K), lambda exonuclease (NEB, M0262L), exonuclease I (NEB, M0293L) and exonuclease III (NEB, M0206L). The circularized DNA was then treated with Quick CIP (NEB, M025L) to dephosphorylate 5 and 3 ends of any residual linear DNA. Circularized genomic DNA (125ng) was treated with ABE8eSpRY:sgRNA-49.3 complexes in vitro in a 50l reaction for 24h at 37C. ABE RNP complexes nicked the targeted DNA strand and deaminated adenine bases to inosine in the non-targeted stranded DNA of both on- and off-target sites. Further enzymatic steps were included with ABE treatment in the CHANGE-seq-BE method to generate double-strand breaks. Nicked DNA circles were treated with endonuclease V in 10 NEB buffer 4 (NEB, M0305S). Endo V cleaved DNA adjacent to inosines to generate linear DNA with 5 overhangs. Gaps were filled with klenow fragments (3>5 exo) and deoxyribonucleotide triphosphates (dNTPs) (NEB, M0212L) in NEB buffer 2. End-repaired DNA products were A-tailed and further ligated with a hairpin adapter using an HTP library preparation kit (Kapa, KK8235), USER treated and amplified by PCR-barcoded universal primers with NEBNext multiplex oligonucleotides for illumina (NEB, E7600S), using Kapa HiFi HotStart uracil master mix. PCR libraries were quantified by quantitative PCR (KAPA Biosystems, KK4824) and sequenced with 151-8-8151 cycles on an Illumina NextSeq 2000 instrument. CHANGE-seq-BE data analyses were performed using open-source software: https://github.com/tsailabSJ/changeseq/tree/dev.

Validation of off-target sites was performed using the rhAmpSeq system from IDT. rhAmpSeq primer panels for targeted amplification were generated using the rhAmpSeq design tool defining the insert size between 150 and 250bp. Applied primer sequences are listed in Supplementary Table 4. A rhAmpSeq CRISPR library was prepared according to the manufacturers instructions and sequenced on an Illumina MiniSeq instrument (MiniSeq high output kit, 300 cycles). Custom python code and open-source bioinformatic tools were used to analyse rhAmpSeq data. First, we generated FASTQ format files by demultiplexing high-throughput-sequencing BCL data files. Next, the reads were processed using CRISPRessoPooled (v.2.0.41) with quantification_window_size 10, quantification_window_center 10, base_editor_output, conversion_nuc_from A, conversion_nuc_to G. The allele frequency table from the output files was used to calculate the AT-to-GC editing frequency. Specifically, the editing frequency for each on- or off-target site was defined as the ratio between the number of reads containing the edited base (that is, G) in a window from positions 4 to 10 of each protospacer (where the GAA PAM is positions 2123) and the total number of reads. To calculate the statistical significance of off-target editing, we applied a method previously described55. In brief, a 2-by-2 contingency table was constructed using the number of edited reads and the number of unedited reads in the treated sample and its corresponding control sample. Next, a 2 test was done. The FDR was calculated using the BenjaminiHochberg method. Significant off-targets were defined on the basis of: first, FDR0.05 and second, the difference in editing frequency between treated and control (1%).

All cancer cell lines (listed in Supplementary Table 5) were cultured in RPMI-1640 (Sigma-Aldrich, R8758) supplemented with 10% heat-inactivated FCS (Gibco Life Technologies) and 2mM GlutaMAX (ThermoFisher Scientific, 35050061) at 37C.

All cell lines were retrovirally transduced with MI-Luciferase-IRES-mCherry (gift from X. Sun; Supplementary Table 1). Cells were then FACS-sorted on the basis of mCherry expression. After expansion, MOLM-14 and OCIAML2 were profiled for short tandem repeats and tested negative for mycoplasma before being frozen until further use. Jurkat and NALM-6 were purchased from ATCC and were therefore not profiled for short tandem repeats.

DF-1 cells were cultured in DMEM high-glucose medium (Sigma-Aldrich, D5796) supplemented with 10% non-heat-inactivated FCS and 2mM GlutaMAX at 39C (Supplementary Table 5).

Leukocyte buffy coats from anonymous healthy human donors were purchased from the blood-donation centre at Basel (Blutspendezentrum SRK beider Basel, BSZ). PBMCs were isolated by density centrifugation using SepMate tubes (StemCell Technologies, 85450) and the density gradient medium Ficoll-Paque (GE Healthcare) according to the manufacturers protocol. Frozen PBMCs were thawed and human primary T cells were then isolated using an EasySep human T cell isolation kit (Stemcell Technologies, 17951) following the manufacturers protocol. T cells were cultured overnight without stimulation at a density of 1.5106 cells per ml in RPMI-1640 medium supplemented with 10% heat-inactivated human serum (AB+, male; purchased from BSZ), 10mM HEPES (Sigma-Aldrich), 2mM GlutaMAX, 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol and 1% MEM non-essential amino acids (all from Gibco Life Technologies). The next day, the human primary T cells were activated with interleukin-2 (IL-2) (150Uml1, proleukin, University Hospital Basel), IL-7 (5ngml1, R&D Systems), IL-15 (5ngm1, R&D Systems) and Dynabead Human T-Activator CD3/CD28 (1:1 beads:cells ratio) (Gibco, 11132D). The activated cells were de-beaded before electroporation.

Leukopaks were purchased from CytoCare and hCD34+ HSPCs were isolated by the LP-34 process using CliniMACS Prodigy (Miltenyi). Isolated hCD34+ HSPCs were thawed and grown in HSPC medium for two days until electroporation (StemSpan SFEM II (StemCell, 09655) supplemented with 100ngml1 human stem cell factor (hSCF) (Miltenyi, 130-096-695), 100ngml1 human FMS-like tyrosine kinase ligand (hFlt3)-ligand (Miltenyi, 130-096-479), 100ngml1 human thrombopoietin (hTPO) (Miltenyi, 130-095-752) and 60ngml1 hIL-3 (Miltenyi, 130-095-069).

K562 cells (2106) were resuspended in buffer T and mixed with 5g BE plasmid (Supplementary Table 1) and 1.5g sgRNA plasmid for co-electroporation using a Neon transfection system (ThermoFisher, MPK10096; 1,450V, 10ms, 3 pulses). To monitor the electroporation efficiency, GFP expression was evaluated 24h after electroporation using an optical microscope. In Extended Data Fig. 1f, BE results are displayed using a custom BE score: log((sum of editing frequencies per condition/number of edited positions per condition)+1).

De-beaded human activated T cells (1106) were resuspended in 100l Lonza supplemented P3 electroporation buffer with 7.5g BE mRNA (1gl1) and 7.5g sgRNA (3.2gl1) (Supplementary Table 2) and electroporated using the 4D-Nucleofector system (Lonza) with program EH-115. Immediately after electroporation, 900l pre-warmed human T-cells medium was added directly in the cuvettes and incubated for 20min at 37C for the T cells to recover. Cells were then transferred in 48-well flat-bottomed plates (Corning, 3548) and the medium was supplemented with 500Uml1 IL-2. The medium was renewed every two days.

hCD34+ HSPCs (1106) were electroporated 48h after thawing with 7.5g BE mRNA (1gl1) and 13.6g sgRNA (3.2gl1) (Supplementary Table 2) with a 1:100 BE:sgRNA molar ratio, following the same protocol as for human T cells but with program CA-137. Electroporated hCD34+ HSPCs were kept in culture at 0.5106 cells per ml in a six-well flat-bottomed plate (Corning, 3516) in HSPC medium supplemented with 100ngml1 hSCF, 100ngml1 hFlt3ligand and 100ngml1 hTPO for in vivo applications and with the addition of 60ngml1 hIL-3 for in vitro assays. The medium was renewed every five days. Edited hCD34+ HSPCs prepared for in vivo injection were frozen two days after electroporation in cryo-preservation CryoStor CS10 medium (Stem Cell Technologies, 07930) at a density of 10106 cells ml1.

The CFU assay was started 72h after gene editing. For each condition, 1.1ml semi-solid methylcellulose medium (StemCell Technologies) containing 200 cells was plated in a well of a SmartDish (StemCell Technologies, 27370) in duplicates. The cells were incubated at 37C, for 14days. The resulting progenitor colonies were counted and scored using STEMVision Analysis (StemCell Technologies) according to the manufacturers instructions. The mean of the total number of colonies in the NTC samples for each experiment was set as 1.

Plasmid (6.5g) encoding WT hCD45RO or its variants was mixed with 200l serum-free DMEM medium and 19.5l polyethylenimine (1mgml1; Chemie Brunschwig, POL23966-100). The transfection mix was added dropwise to 1106 DF-1 cells plated the day before in a six-well plate. Cells were analysed 48h later by flow cytometry.

For in vitro ADC killing assays, 5,000 base-edited human activated T cells or 25,000 base-edited hCD34+ HSPCs were plated five days after electroporation in 96-well plates (flat-bottomed for T cells and round-bottomed for HSPCs; Corning 3596 and 3799, respectively) in 100l of corresponding medium (supplemented with only 50Uml1 of IL-2 for human T cells). For ADC killing assays involving saporin, a 100nM stock was prepared by incubating the biotinylated antibody (BC8 or MIRG451 mAbs) and saporinstreptavidin (ATS-Bio, IT-27-1000) at a 1:1 molar ratio for 30min at room temperature.

For in vitro ADC killing of co-cultures, 12,500 Jurkat cells were stained for 20min with CTV (Invitrogen, C34557A) at 37C and then seeded at a 1:1 cell ratio with 12,500 base-edited hCD34+ HSPCs five days post-electroporation in 96-well round-bottom plates in 100l HSPC medium with corresponding concentrations of CIM053SG3376 (ADC Therapeutics). Cells were incubated for 72h at 37C, stained for flow cytometry or cell sorting and analysed using a BD LSRFortessa. Genomic DNA was extracted for sequencing.

For in vitro ADC killing of mCherryluciferase-marked tumour cell lines (Jurkat, NALM-6, OCI-AML-2 and MOLM-14), 2,000 cells were plated in 384-well plates in medium with or without 30min pre-incubation at 37C with 50gml1 (333.33nM) naked CIM053 antibody (40l final total volume per well). Following a 72h incubation period, 5l firefly d-luciferin (0.75mgml1 resuspended in medium (Biosynth, L-8220)) was added to each well and incubated for 5min at room temperature. Luminescence readouts were recorded using a BioTek Synergy H1 plate reader.

For precise antibodyprotein affinity measurements and biophysical characterization, CD45wt and variants containing only D1 and D2 of the ECD were produced. The protein sequence (residues 225394) was histidine tagged at the carboxy terminus and contains few N- and C-terminal added amino acids (full WT sequence: ETGIEGRKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEGTKHHHHHH, SEQ ID 57, Uniprot ID P08575). Expi293F GnTI cells (Thermo Fisher, A39240) that lack N-acetylglucosaminyltransferase I (GnTI) activity and therefore lack complex N-glycans were used for protein expression. After collection, the protein was purified using Ni-NTA chromatography followed by digestion of high-mannose glycans with endoglycosidase H (EndoHf; New England BioLabs, P0703S) at 37C overnight. EndoHf was removed from the protein solution with amylose resin and the CD45 protein was further purified by size-exclusion chromatography in buffer comprising 20mM HEPES, pH 7.4, 150mM NaCl. Peak monomer and dimer fractions (where needed) were concentrated using a 10kDa cut-off Amicon centrifugal filter (UFC8010) and protein aliquots were flash-frozen in liquid nitrogen before storage at 150C. Variant CD45 proteins were produced using the same experimental procedure. The monomer content percentage for each protein was taken from the size-exclusion chromatogram.

Analysis of MIRG451 and BC8 binding to the selected variants was performed on an Octet system RED96e (Sartorius) or R8 (Sartorius) at 25C with shaking at 1,000 rpm using 1 kinetic buffer (Sartorius, 18-1105). The selected variants were screened for their ability to bind to MIRG451 and BC8 using different concentrations of CD45 (WT or variant). MIRG451 was captured by an anti-human Fc-capture biosensor (AHC) (Sartorius, 18-5060) for 300s at 0.51gml1. As an analyte, human CD45wt and variants, containing only domains 1 and 2, were titrated at seven different concentrations, from 1,000nM to 15.6nM or from 50nM to 0.78nM with a 1:2 dilution series. Association of the analyte to MIRG451 was monitored for 600s and dissociation of the analyte from MIRG451 was monitored for 1,800s. Reference subtraction was performed against buffer-only wells. AHC tips were regenerated using 10mM Gly-HCl, pH 1.7. Data were analysed using the Octet data analysis software HT 12.0. Data were fitted to a 1:1 binding model. Kinetic rates Ka and Kd were globally fitted.

To analyse binding to BC8, streptavidin biosensors (Sartorius, 18-5020) were first coated with CaptureSelect biotin anti-LC- (murine) conjugate (Thermo Scientific, 7103152100) for 600s at 1gml1. BC8 was then captured by the coated streptavidin biosensors for 300s at 0.51.0gml1. Analyte titration was performed as for MIRG451. Association of the analyte to BC8 was monitored for 300s and dissociation of the analyte from BC8 was monitored for 900s. Reference subtraction, regeneration and data analysis were performed as for MIRG451.

The thermostability of CD45 D1D2 variants was analysed by differential scanning fluorimetry and monitoring tryptophane fluorescence using Nanotemper Prometheus NT.48 NanoDSF or a Nanotemper Prometheus Panta (NanoTemper Technologies)56,57,58. Thermal denaturation was monitored by tryptophane/tyrosine fluorescence at 350 and 330nm and an excitation wavelength of 280nm was used. CD45wt and variants were prepared at 0.251.0mgml1 in 20mM HEPES, 150mM NaCl, pH 7.4. Then 10l was put into the capillaries and placed into the sample holder. Each protein was measured in triplicates per experiment and the CD45wt was measured in four different experiments. The temperature was increased from 20C to 90C or 95C. The analysis was performed using the ratio of the fluorescent intensities at 350 and 330nm. The software of the instrument was used to calculate Tonset and TM as well as the mean and s.d. of the triplicates. The melting temperature was determined as the inflexion point of the sigmoidal curve and compared with that of CD45wt.

Flow cytometry was done on BD LSRFortessa instruments with BD FACSDiva software. Data were analysed with FlowJo software. Antibodies used for flow cytometry are listed in Supplementary Table 6. Cells were sorted with BD FACSAria or BD FACSMelody cell sorter instruments. Sorted cells were resuspended in 30l QuickExtract. PCRs were performed and sent for Sanger sequencing.

The CD45 expression of 27 people diagnosed with AML at University Hospital Basel was assessed using routinely acquired flowcytometry data as part of the diagnostic work-up. Gating for AML blasts, lymphocytes and erythrocytes was performed manually using FlowJo 10.10.0. Owing to the experimental set-up (threshold for SSC-A and FSC-A to exclude debris), a distinct erythrocyte population could not be distinguished in all samples (23 of 27). Data were analysed with GraphPad Prism 10 and statistical significance was calculated using mixed-effects analysis. All patients gave written informed consent to the analysis of clinical data for research purposes and the study was approved by the local ethics committee (BASEC-Nr 2023-01372).

All animal work was done in accordance with the federal and cantonal laws of Switzerland. Protocols were approved by the Animal Research Commission of the Canton of Basel-Stadt, Switzerland. All mice were housed in a specific pathogen-free condition in accordance with institutional guidelines and ethical regulations. NBSGW (stock 026622) female mice were purchased from Jackson Laboratories. HSPCs were edited as described above. Two days after electroporation, cells were collected and frozen in CryoStor CS10 medium. Cells were thawed on the day of injection, washed and resuspended in PBS. Recipient NBSGW female mice (4 weeks old) were injected intravenously into the tail vein with HSPCs (the number of cells injected varied between 0.6 and 1.1 million and is indicated in each figure). Chimerism was analysed by flow cytometry in blood after ten weeks. Mice were treated with saline or CIM053SG3376 at the dose(s) and intervals indicated in each figure. For tumour experiments in humanized mice, 1106 MOLM-14mCherryluc cells were injected into the tail vein. Then, 10 or 12 days after tumour inoculation, the mice were treated with saline or 1mg per kg CIM053SG3376. The mice received a second antibody dose of 0.5mg per kg CIM053SG3376 10 or 25 days after the first dose. Mice were euthanized 43 or 45 days after tumour inoculation or when reaching the maximum allowed clinical score. To monitor tumour growth, mice were injected intraperitoneally with 100l d-luciferin (BioSynth, L-8220) and were subjected to Newton7.0 imaging (Vilber).

For secondary transplant, NSGSGM3 female mice (stock 013062) were purchased from Jackson Laboratories. Recipient mice were irradiated the day before the BM transplant with 200cGy. Primary transplant mice were euthanized, the BM was isolated and 40% of it was re-injected into the new host. Mice from secondary transplants were euthanized 8 weeks after humanization.

MOLM-14mCherryluc (1106), OCI-AML-2mCherryluc (2106), JurkatmCherryluc (5106) or NALM-6mCherryluc (0.5106) cells were injected into the tail vein of NBSGW mice. After tumour inoculation, mice were monitored regularly (for behaviour, weight and imaging). Mice were treated with saline, control-SG3376 or CIM053SG3376 at the dose and intervals indicated in the relevant figure and euthanized 21 days after tumour inoculation or when reaching the maximum allowed clinical score.

Deidentified patient-derived AML samples were obtained from the PDX repository59,60 (Cancer Research Center of Toulouse, France). Signed written informed consent for research use in accordance with the Declaration of Helsinki was obtained from patients and approved by the Geneva Health Department Ethic Committee. PDX cells (0.6106) were injected into the tail vein of humanized NBSGW mice (8 weeks after HSPC injection). The weight of the mice was monitored regularly. Mice were treated with saline or CIM053SG3376 at the doses and intervals indicated in the figure and the mice were euthanized 54 days after tumour inoculation. Some control mice were euthanized 3 days before antibody treatment.

After the mice were euthanized, 0.2ml blood, both hind legs (femur and tibia) and the spleen were collected from each mouse. Cell suspensions were generated, red blood cells were lysed using ACK lysis buffer and then the cell suspensions were filtered. For tumour experiments, organs were collected on the day of euthanasia, single cell suspensions were generated and frozen in cryo medium. Samples from all mice were thawed and stained on the same day to minimize experimental variability. Cells were stained for different antigens and 30l Accucheck counting beads (1,066 microspheres per l; Invitrogen, PCB100) were added to each sample and the results were analysed by FACS using a BD LSRFortessa instrument.

Statistical analyses were done using GraphPad Prism 9 and 10 software. In all figure legends, n refers to the number of experimental replicates. For multiple comparisons, two-way ANOVA tests were used with significance levels indicated. Data are presented as meanstandard deviation. Survival curves were analysed using the log-rank MantelCox test. rhAmpSeq was analysed using a 2 test. The FDR was calculated using the BenjaminiHochberg method.

Some data points of the in vivo experiments were excluded after visual inspection of samples if the FACS time gate showed irregularities. One mouse that did not engraft HSPCs was excluded from Fig. 5 and Extended Data Fig. 10. Cell numbers in the sgNTC group treated with CIM053SG3376 were so low that analysis of some assays became unreliable (NGS, genetic chimerism analysis). We therefore excluded this group from NGS.

The number of biological replicates is specified for each experiment in the relevant figure legend. Several key experiments were performed by different people at times in different laboratories, and reagents were shared. For instance, identification of variants, characterization of recombinant variants and FACS validation were performed by different people. Some experiments were performed in the academic lab and validated in Cimeio labs and vice versa. To avoid unconscious bias when assigning mice to saline or the CIM053SG3376 groups, we always assigned the mice with the largest tumour mass to the ADC group.

The investigator who determined genetic chimerism (NGS and analysis) was blinded and provided the results to the investigator in charge of supervising in vivo experiments. The people who performed CHANGE-Seq_BE, rhAMPSeq and analysed the data were blinded and provided the results to the investigator in charge of supervising the in vivo experiments.

Non-proprietary materials are freely available on reasonable request. Restrictions apply to proprietary, commercial material.

Further information on research design is available in theNature Portfolio Reporting Summary linked to this article.

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Treating neurons and brain organoids with a modified form of CRISPR rescues the effects of pathogenic variants in two high-confidence autism-linked genes, according to a new preprint. The approach boosts the expression of the genes, CHD8 and SCN2A, by targeting structures that regulate them.

SCN2A codes for an ion channel that helps propagate electrical signals across the brain. Variants in that gene are associated with seizures, autism and intellectual disability. CHD8 is involved in the remodeling of chromatin, the complex of DNA and proteins that makes up chromosomes. People with faulty copies of CHD8 typically have autism and a larger-than-average head.

The work suggests that the impact of autism-related variants could be reversed by altering gene expression, as opposed to altering genes directly, although more research is needed before the technique can find applications in the clinic, experts say.

This is another way in which we can regulate genes that are extremely important in [autism], so thats a major finding, says Kevin Bender, associate professor of neurology at the University of California, San Francisco, who was not involved in the study. But its really the first step in understanding whether this approach is broadly applicable.

In the past, Bender and his colleagues used the same version of CRISPR, which activates genes rather than editing them, to boost the expression of SCN2A in mice with a harmful variant in one copy of the gene. The treatment corrected problems in the animals neurons.

By increasing the expression of SCN2A in mutant neurons and brain organoids, the new study confirmed many of the previous findings. But the work also revealed, for the first time, that activating gene regulatory elements with CRISPR could counteract the effects associated with harmful variants in CHD8.

Because that gene controls the expression of thousands of other genes, to see that the upregulation of CHD8 is restorative in some way is really exciting, Bender says.

T

So Geschwind and his team used CRISPR to boost activity in noncoding regions of the genome called enhancers, which can regulate a genes transcription, in this case linked to SCN2A and CHD8.

Boosting enhancers of CHD8 in neurons and brain organoids that lacked a functional copy of that gene led to a reduction in organoid size and in the number of differentially expressed genes. This finding suggests the intervention can rescue cells: without it, CHD8 mutant organoids are larger and show an over-proliferation of neural progenitor cells in comparison with controls that have two functional gene copies.

To see that the upregulation of CHD8 is restorative in some way is really exciting.

Similarly, enhancing the expression of SCN2A reversed the problems observed in neurons and brain organoids that lack the gene, including impaired development, reduced excitability and sluggish responses to electrical currents.

The team posted their findings on the preprint server bioRxiv in March.

Identifying enhancers for SCN2A and CHD8 was a feat in itself, Bender says. Enhancers arent typically located close to the genes they regulate. To find them, scientists must undertake a treasure hunt, he says. Their ability to do that was really remarkableand theyve done it for two major [autism-linked] genes.

Unlike traditional CRISPR approaches, which cut DNA to delete or insert variants and can have off-target effects, CRISPR activation may be less likely to cause harm. But before the approach finds its way to the clinic, researchers will need to determine its safety profile and look for any unintended consequences, saysNadav Ahituv, professor of bioengineering and therapeutic sciences at the University of California, San Francisco, who was not involved in the study.

Ahituv, who has worked with Bender on activating SCN2A using CRISPR, is co-founder of a company that is developing CRISPR therapies that target SCN2A and SCN1A, which has also been linked to autism.

Researchers must also identify a suitable way to deliver the intervention to the brain, Geschwind says. People are working out in-utero gene delivery, so I dont think its that far off in the next decade, he says. Im fairly optimistic about this type of approach.

The new study, he adds, also avoids the need to invest significant resources and time into deciphering the functions and mechanisms of individual genes, as well as developing methods to counteract them. Instead, it uses a genes regulatory elements to activate its expression. To me, thats an exciting therapeutic shortcut.

Read the original post:
CRISPR gives autism-linked genes a boost, rescues functioning - The Transmitter: Neuroscience News and Perspectives

Plasmid design and construction

A summary of plasmid constructs are in Supplementary Table 1 and plasmid sequences are in Supplementary Data 2. Unless otherwise specified, cloning was performed by Gibson Assembly of PCR-amplified or commercially synthesized gene fragments (from Integrated DNA Technologies or Twist Bioscience) using NEBuilder Hifi Master Mix (NEB, E262), and final plasmids sequence-verified by Sanger sequencing of the open reading frame and/or commercial whole-plasmid sequencing service provided by Primordium.

To summarize, denAsCas12a-KRAB, multiAsCas12a-KRAB, multiAsCas12a and enAsCas12a-KRAB open reading frames were embedded in the same fusion protein architecture consisting of an N-terminal 6xMyc-NLS29 and C-terminal XTEN80-KRAB-P2A-BFP103. The denAsCas12a open reading frame was PCR amplified from pCAG-denAsCas12a(E174R/S542R/K548R/D908A)-NLS(nuc)-3xHA-VPR (RTW776) (Addgene, plasmid 107943 (ref. 30)). AsCas12a variants described were generated by using the denAsCas12a open reading frame as starting template and introducing the specific mutations encoded in overhangs on PCR primers that serve as junctions of Gibson assembly reactions. opAsCas12a (ref. 29) is available as Addgene plasmid 149723, pRG232. 6xMyc-NLS was PCR amplified from pRG232. KRAB domain sequence from KOX1 was previously reported42. The lentiviral backbone for expressing Cas12a fusion protein constructs expresses the transgene from an SFFV promoter adjacent to UCOE and is a gift from Marco Jost and Jonathan Weissman, derived from a plasmid available as Addgene 188765. XTEN80 linker sequence was taken from a previous study51 and was originally from Schellenberger et al.111. For constructs used in piggyBac transposition, the open reading frame was cloned into a piggyBac vector backbone (Addgene, 133568) and expressed from a CAG promoter. Super PiggyBac Transposase (PB210PA-1) was purchased from System Biosciences.

dAsCas12a-3xKRAB open reading frame sequence is from a construct originally referred to as SiT-ddCas12a-[Repr]27. We generated SiT-ddCas12a-[Repr] by introducing the DNase-inactivating E993A by PCR-based mutagenesis using SiT-Cas12a-[Repr] (Addgene, 133568) as template. Using Gibson Assembly of PCR products, we inserted the resulting ddCas12a-[Repr] open reading frame in-frame with P2A-BFP in a piggyBac vector (Addgene, 133568) to enable direct comparison with other fusion protein constructs cloned in the same vector backbone (crRNAs are encoded on separate plasmids as described below).

Fusion protein constructs described in Supplementary Fig. 8bf were assembled by subcloning the protein-coding sequences of AsCas12a and KRAB into a lentiviral expression vector using the In-Fusion HD Cloning system (TBUSA). AsCas12a mutants were cloned by mutagenesis PCR on the complete wild-type AsCas12a vector to generate the final lentiviral expression vector.

All individually cloned crRNA constructs and their expression vector backbone are listed in Supplementary Table 1. Unless otherwise specified, individual single and 3-plex crRNA constructs were cloned into the human U6 promoter-driven expression vector pRG212 (Addgene, 149722 (ref. 29)), which contains wildtype (WT) direct repeats (DR). Library 1, Library 2, and some 3-plex and all 4-plex, 5-plex and 6-plex As. crRNA constructs were cloned into pCH67, which is derived from pRG212 by replacing the 3 DR with the variant DR8 (ref. 28). For constructs cloned into pCH67, the specific As. DR variants were assigned to each position of the array as follows, in 5 to 3 order:

3-plex: WT DR, DR1, DR3, DR8

4-plex: WT DR, DR1, DR10, DR3, DR8

5-plex: WT DR, DR1, DR16, DR10, DR3, DR8

6-plex: WT DR, DR1, DR16, DR18, DR10, DR3, DR8

8-plex: WT DR, DR1, DR16, DR_NS1, DR17, DR18, DR10, DR3, DR8

10-plex: WT DR, DR1, DR16, DR_NS1, DR4, DR_NS2, DR17, DR18, DR10, DR3, DR8

DR sequences are as follows: WT DR=AATTTCTACTCTTGTAGAT, DR1=AATTTCTACTGTCGTAGAT, DR16=AATTCCTACTATTGTAGGT, DR_NS1=AATTCCTCCTCTTGGAGGT, DR4=AATTTCTACTATTGTAGAT, DR_NS2=AATTCCTCCTATAGGAGGT, DR17=AATTTCTCCTATAGGAGAT, DR18=AATTCCTACTCTAGTAGGT, DR10=AATTCCTACTCTCGTAGGT, DR3=AATTTCTACTCTAGTAGAT, DR8=AATTTCTCCTCTAGGAGAT. Sequences for DR variants were previously reported28, except for DR_NS1 and DR_NS2, which were newly designed based on combining previously reported variants28. The rationale for selecting specific DR variants was to minimize homology across variants and maintain high crRNA activity based on prior analysis28.

1-plex,3-plex, 8-plex, and 10-plex crRNA constructs were cloned by annealing sets of complementary oligos with compatible overhangs in spacer regions, phosphorylation by T4 polynucleotide kinase (NEB M0201S), and ligated with T4 DNA ligase (NEB M0202) into BsmbI site of vector backbones. 4-plex, 5-plex and 6-plex crRNA arrays were ordered as double-stranded gene fragments and cloned into the BsmbI site of vector backbones by Gibson Assembly using the NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621). Functions for designing oligos or gene blocks for cloning crRNA arrays are available as an R package at https://github.com/chris-hsiung/bears01.

All spacer and PAM sequences are provided in Supplementary Table 1. For cloning individual crRNA constructs targeting TSSs, CRISPick (https://portals.broadinstitute.org/gppx/crispick/public) was used in the enAsCas12a CRISPRi mode (by providing gene name) or CRISPRko mode (by providing sequence for TSS-proximal regions) to design spacers targeting canonical (TTTV) or non-canonical PAMs generally located within 50-bp to +300-bp region around the targeted TSS whenever possible, but some sites farther from the annotated TSS can show successful CRISPRi activity and were used. We manually selected spacers from the CRISPick output by prioritizing the highest on-target efficacy scores while avoiding spacers with high off-target predictions. The same non-targeting spacer was used throughout the individual well-based experiments and was randomly generated and checked for absence of alignment to the human genome by BLAT112.

The hg19 genomic coordinates for MYC enhancers are e1 chr8:128910869-128911521, e2 chr8:128972341-128973219 and e3 chr8:129057272-129057795. DNA sequences from those regions were downloaded from the UCSC Genome Browser and submitted to CRISPick. The top three spacers targeting each enhancer were picked based on CRISPick on-target efficacy score, having no Tier I or Tier II Bin I predicted off-target sites, and considering proximity to peaks of ENCODE110 DNase hypersensitivity signal (UCSC Genome Browser113 accession # wgEncodeEH000484, wgEncodeUwDnaseK562RawRep1.bigWig) and H3K27Ac ChIP-seq signal (UCSC Genome Browser accession # wgEncodeEH000043, wgEncodeBroadHistoneK562H3k27acStdSig.bigWig). These DNase hypersensitivity and H3K27Ac ChIP-seq tracks were similarly used to nominate candidate enhancer regions at the CD55 locus, whose genomic sequences are provided in Supplementary Table 1.

C4-2B cells114 were gifted by F. Feng, originally gifted by L. Chung. All cell lines were cultured at 37C with 5% CO2 in tissue culture incubators. K562 and C4-2B cells were maintained in RPMI-1640 (Gibco, 22400121) containing 25mM HEPES, 2 mM L-glutamine and supplemented with 10% FBS (VWR), 100 U ml1 streptomycin, and 100mg ml1 penicillin. For pooled screens using K562 cells cultured in flasks in a shaking incubator, the culture medium was supplemented with 0.1% Pluronic F-127 (Thermo Fisher, P6866). HEK 293T cells were cultured in media consisting of DMEM, high glucose (Gibco 11965084, containing 4.5g ml1 glucose and 4mM L-glutamine) supplemented with 10% FBS (VWR) and 100units/mL streptomycin, 100mg ml1 penicillin. Adherent cells were routinely passaged and harvested by incubation with 0.25% trypsin-EDTA (Thermo Fisher, 25200056) at 37C for 510min, followed by neutralization with media containing 10% FBS.

Unless otherwise specified below, lentiviral particles were produced by transfecting standard packaging vectors (pMD2.G and pCMV-dR8.91) into HEK293T using TransIT-LT1 Transfection Reagent (Mirus, MIR2306). At <24h after transfection, culture medium was exchanged with fresh medium supplemented with ViralBoost (Alstem Bio, VB100) at 1:500 dilution. Viral supernatants were harvested ~4872h after transfection and filtered through a 0.45mm PVDF syringe filter and either stored in 4C for use within <2 weeks or stored in 80C until use. Lentiviral infections included polybrene (8g/ml). MOI was estimated from the fraction of transduced cells (based on fluorescence marker positivity) by the following equation115,116: MOI=ln(1 fraction of cells transduced).

For experiments described in Supplemental Fig. 8af, lentivirus was produced by transfecting HEK293T cells with lentiviral vector, VSVG and psPAX2 helper plasmids using polyethylenimine. Medium was changed ~68h post transfection. Viral supernatant was collected every 12h five times and passed through 0.45-m PVDF filters. Lentivirus was added to target cell lines with 8g ml1 polybrene and centrifuged at 650g for 25min at room temperature. Medium was replaced 15h after infection. An antibiotic (1g ml1 puromycin) was added 48h after infection.

For piggyBac transposition of fusion protein constructs, cells were electroporated with ~210ng of AsCas12a fusion protein plasmid and ~84ng of Super PiggyBac Transposase Expression Vector (PB210PA-1, Systems Biosciences) using the SF Cell Line 4D-Nucleofector X Kit (V4XC-2032, Lonza Bioscience) and the 4D-Nucleofector X Unit as per manufacturers instructions (FF-120 program for K562 cells; EN-120 program for C4-2B cells).

The following antibodies were used for flow cytometry at 1:100 dilution: CD55-APC (BioLegend, 311312), CD55-PE (BioLegend, 311308), CD81-PE (BioLegend, 349506), CD81-AlexaFluor700 (BioLegend, 349518), B2M-APC (BioLegend, 316311), KIT-PE (BioLegend, 313204), KIT-BrilliantViolet785 (BioLegend, 313238) and FOLH1-APC (BioLegend, 342508). Cells were stained with antibodies were diluted in FACS Buffer (PBS with 1% BSA) and washed with FACS Buffer, followed by data acquisition on the Attune NxT instrument in 96-well plate format unless otherwise specified. For CRISPRi experiments, all data points shown in figures are events first gated for single cells based on FSC/SSC, then gated on GFP-positivity as a marker for cells successfully transduced with crRNA construct, as exemplified in Supplementary Fig. 1. For CRISPRi experiments in C4-2B cells, propensity score matching on BFP signal was performed using the MatchIt v4.5.3R package.

For cell fitness competition assays, the percentage of cells expressing the GFP marker encoded on the crRNA expression vector is quantified by flow cytometry. log2 fold-change of percentage of GFP-positive cells was calculated relative to day 2 (for experiments targeting the Rpa3 locus in Supplementary Fig. 8) or day 6 (for experiments targeting the MYC locus in Fig. 6b). For experiments targeting the Rpa3 locus, flow cytometry was performed on the Guava Easycyte 10 HT instrument.

For all crRNAs in Library 1 and Library 2, we excluded in the analysis spacers with the following off-target prediction criteria using CRISPick run in the CRISPRi setting: 1) off-target match = MAX for any tier or bin, or 2) # Off-Target Tier I Match Bin I Matches > 1). The only crRNAs for which this filter was not applied are the non-targeting negative control spacers, which do not have an associated CRISPick output. All crRNA sequences were also filtered to exclude BsmbI sites used for cloning and three or more consecutive Ts, which mimic RNA Pol III termination signal.

To design crRNA spacers targeting gene TSSs for Library 1, we used the 50-bp to +300-bp regions of TSS annotations derived from capped analysis of gene expression data and can include multiple TSSs per gene67. We targeted the TSSs of 559 common essential genes from DepMap with the strongest cell fitness defects in K562 cells based on prior dCas9-KRAB CRISPRi screen67. We used CRISPick with enAsCas12a settings to target all possible PAMs (TTTV and 44 non-canonical PAMs) in these TSS-proximal regions. Except for the criteria mentioned in the previous paragraph, no other exclusion criteria were applied. For the TSS-level analyses shown in Fig. 4d,e, each gene was assigned to a single TSS targeted by the crRNA with the strongest fitness score for that gene.

Negative controls in Library 1 fall into two categories: 1) 524 intergenic negative controls, and 2) 445 non-targeting negative controls that do not map to the human genome. Target sites for intergenic negative controls were picked by removing all regions in the hg19 genome that are within 10kb of annotated ensembl genes (retrieved from biomaRt from https://grch37.ensembl.org) or within 3kb of any ENCODE DNase hypersensitive site (wgEncodeRegDnaseClusteredV3.bed from http://hgdownload.cse.ucsc.edu/goldenpath/hg19/encodeDCC/wgEncodeRegDnaseClustered/). The remaining regions were divided into 1-kb fragments. 90 such 1-kb fragments were sampled from each chromosome. Fragments containing 20 consecutive Ns were removed. The remaining sequences were submitted to CRISPick run under CRISPRi settings. The CRISPick output was further filtered for spacers that meet these criteria: 1) off-target prediction criteria described in the beginning of this section, and 2) on-target Efficacy Score 0.5 (the rationale is to maximize representation by likely active crRNAs to bias for revealing any potential cell fitness effects from nonspecific genotoxicity due to residual DNA cutting by multiCas12a-KRAB), 3) mapping uniquely to the hg19 genome by Bowtie117 using -m 1 and otherwise default parameters, 3) filtered once more against those whose uniquely mapped site falls within 10kb of annotated ensembl genes or any ENCODE DNase hypersensitive site.

Non-targeting negative control spacers were generated by 1) combining non-targeting negative controls in the Humagne C and D libraries (Addgene accession numbers 172650 and 172651), 2) taking 20-nt non-targeting spacers from the dCas9-KRAB CRISPRi_v2 genome-wide library67, removing the G in the 1st position and appending random 4-mers to the 3 end. This set of spacers were then filtered for those that do not map to the hg19 genome using Bowtie with default settings.

Sublibrary A (42,600 constructs designed): Test position spacers were encoded at each position of the 6-plex array, with remaining positions referred to as context positions and filled with negative control spacers. Test positions encodes one of 506 intergenic negative control spacers and 914 essential TSS-targeting spacers. The essential TSS-targeting spacers were selected from among all spacers targeting PAMs within 50-bp to +300-bp TSS-proximal regions of 50 common essential genes with the strongest K562 cell fitness defect in prior dCas9-KRAB CRISPRi screen67 and must have 0.7 CRISPick on-target efficacy score. Negative control context spacers consist of five 6-plex combinations; three of these combinations consist entirely of non-targeting negative controls, and two of the combinations consist entirely of intergenic negative controls.

Sublibrary B (6,370 constructs designed): crRNA combinations targeting cis-regulatory elements at the MYC locus were assembled from a subset of combinations possible from 15 starting spacers (3 targeting MYC TSS, 3 targeting each of 3 enhancers, and 3 intergenic negative control spacers). The three enhancer elements are described in the subsection Design of individual crRNAs. These 15 starting spacers were grouped into 5 3-plex combinations, each 3-plex combination exclusively targeting one of the four cis-regulatory elements, or consisting entirely of intergenic negative controls. Each 3-plex was then encoded in positions 13 of 6-plex arrays, and positions 46 were filled with all possible 3-plex combinations chosen from the starting 15 spacers. All 6-plex combinations were also encoded in the reverse order in the array.

All-negative control constructs (2,000 constructs designed): 1,500 6-plex combinations were randomly sampled from the intergenic negative control spacers described for Library 1. 500 6-plex combinations were randomly sampled from non-targeting negative control spacers described for Library 1.

Intergenic negative controls and non-targeting negative controls are defined the same as in Library 1.

As Library 2 was designed and cloned prior to the completion of the Library 1 screen, the majority of Library 2 contains constructs encoding for spacers in the test position that in hindsight do not produce strong phenotypes as single crRNAs in the Library 1 screen.

Both Library 1 and Library 2 were constructed from pooled oligonucleotide libraries designed to contain crRNA constructs designed for exploratory analysis for a separate unpublished study. Sequencing reads from those non-contributory constructs are present in the raw fastq files, do not affect interpretation of Library 1 and Library 2 screen cell fitness scores, and are excluded from analysis in the present study.

All PCRs were performed with NEBNext Ultra II Q5 Master Mix (NEB M0544). For Library 1, ~140 fmol pooled oligo libraries from Twist were subjected to 10 cycles of PCR amplification using primers specific to adaptor sequences flanking the oligos and containing BsmbI sites. The PCR amplicons were cloned into a crRNA expression backbone (pCH67) by Golden Gate Assembly with ~1:1 insert:backbone ratio using ~500 fmol, followed by bacterial transformation to arrive at an estimated 778 coverage in the final plasmid Library 1. For Library 2, 915 fmol of pooled oligo libraries from Twist was subjected to 18 cycles of PCR amplification and agarose gel purification of the correctly sized band before proceeding to Golden Gate Assembly. The estimated coverage of plasmid Library 2 from bacterial colony forming units is ~60. Additional details are described in Supplementary Information.

Primer sequences are provided in Supplementary Table 2. Sequences of the expected PCR amplicons for Illumina sequencing are in Supplementary Data 2. crRNA inserts were amplified from genomic DNA isolated from screens using 16 cycles of first round PCR using pooled 0-8nt staggered forward and reverse primers, treated with ExoSAP-IT (Thermo Fisher, 78201.1.ML), followed by 7 cycles of round 2 PCR to introduce Illumina unique dual indices and adaptors. Sequencing primer binding sites, unique dual indices, P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document #1000000002694 v16. PCR amplicons were subject to size selection by magnetic beads (SPRIselect, Beckman, B23318) prior to sequencing on an Illumina NovaSeq6000 using SP100 kit (PE100) for Library 1 or SP500 kit (PE250) for Library 2. Sequencing of plasmid libraries were performed similarly, except 7 cycles of amplification were each used for Round 1 and Round 2 PCR. The size distribution of the final library was measured on an Agilent TapeStation system. We noted that even after magnetic bead selection of Round 2 PCR-amplified Library 2 plasmid library (colonies from which were Sanger sequencing verified) and genomic DNA from screens, smaller sized fragments from PCR amplification during Illumina sequencing library preparation persisted. Thus, the majority of unmapped reads likely reflect undesired PCR by-products, though lentiviral recombination could contribute at an uncertain but relatively low frequency as well.

Library 1 screen: K562 cells engineered by piggyBac transposition to constitutively express denAsCas12a-KRAB or multiAsCas12a-KRAB were transduced with lentivirally packaged Library 1 constructs at MOI ~0.15. Transduced cells were then selected using 1g/ml puromycin for 2 days, followed by washout of puromycin. On Day 6 after transduction, initial (T0) time point was harvested, and the culture was split into 2 replicates that are separately cultured henceforth. 10 days later (T10), the final time point was harvested (8.6 total doublings for multiAsCas12a-KRAB cells, 9.15 total doublings for denasCas12a-KRAB cells). A cell coverage of >500 was maintained throughout the screen. Library 2 screen: K562 cells engineered by piggyBac transposition to constitutively express multiAsCas12a-KRAB were transduced with lentivirally packaged Library 2 constructs at MOI ~0.15. The screen was carried out similarly as described for Library 1 screen, except the screen was carried out for 14 days (T14) or 13.5 total doublings and maintained at a cell coverage of >2,000 throughout. Genomic DNA was isolated using the NucleoSpin Blood XL Maxi kit (Machery-Nagel, 740950.50).

Summary of library contents are in Supplementary Fig. 18.

For Library 1, reads were mapped to crRNA constructs using sgcount (https://noamteyssier.github.io/sgcount/), requiring perfect match to the reference sequence. For Library 2, reads were mapped using an algorithm (detailed in Supplementary Information) requiring perfect match to the reference sequence, implemented as casmap constructs command in a package written in Rust, available at https://github.com/noamteyssier/casmap.

Starting from read counts, the remainder of analyses were performed using custom scripts in R. Constructs that contained less than 1 reads per million (RPM) aligned to the reference library in either replicates at T0 were removed from analysis. From the constructs that meet this read coverage threshold, a pseudocount of 1 was added for each construct and the RPM recalculated and used to obtain a fitness score118 that can be interpreted as the fractional defect in cell fitness per cell population doubling:

$${gamma }=log_2left(frac{left({mathrm{RPMfinal}}/{mathrm{negctrlmedianRPMfinal}}right)}{left({mathrm{RPMinitial}}/{mathrm{negctrlmedianRPMinitial}}right)}right)Big/{mathrm{totaldoublings}},$$

where RPM is the read count per million reads mapped to reference (initial = at T0, final = at end of screen), negctrlmedian is the median of RPM of intergenic negative control constructs, totaldoublings is the total cell population doublings in the screen. For Library 1, data from a single T0 sample was used to calculate the fitness score for both replicates due to an unexpected global loss of sequencing read counts for one of two originally intended T0 replicate samples. For each screen replicate in Library 2, data from two separate sequencing library preps from the same Round 1 PCR material subjected to separate Round 2 PCRs and sequenced on separate runs were pooled together for analysis.

K562 cell lines engineered with the corresponding Cas12a protein constructs were transduced with crRNAs and sorted for transduced cells based on GFP-positivity. 200,000 cells were collected 14 or 15 days after crRNA transduction and genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, 740951.50). For analysis of CD55 and CD81 loci, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial Illumina adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, 740609.250). Paired-end (2 250bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). Raw fastq files were obtained from GENEWIZ and aligned to reference sequences using CRISPResso2 (ref. 119). Quantification diagrams were generated in R. For analysis at the KIT locus, cells were lysed using QuickExtract DNA Solution (Lucigen) and amplicons were generated using 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher, 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq at the Arc Institute Multi-Omics Technology Center. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document #1000000002694 v16. Bioinformatic analysis of indel frequencies and simulation of indel impacts on gene expression, accounting for DNA copy number of the target region in the K562 genome65, are detailed in Supplementary Information. Primer sequences are in Supplementary Table 2.

Genomic DNA was harvested from 20 million cells using the Qiagen Genomic Tips Kit (10243). As detailed in Supplementary Information, we used a custom protocol adapted from the Nanopore Cas9 Sequencing Kit users manual (SQK-CS9109, though this kit was not actually used) to enrich for genomic DNA surrounding crRNA target sites for Nanopore sequencing using Kit 14 chemistry. Cas9 guide spacer sequences are in Supplementary Table 1.

fastq files generated by MinKNOW version 23.07.15 (Oxford Nanopore Technologies) were aligned to the ~20-kb regions (defined by the outermost Cas9 sgRNA protospacer sites flanking each targeted locus) surrounding each crRNA target site in MinKNOW to generate bam files. Bam files for each sample were merged using samtool merge (samtools v1.6 (ref. 120)). Merged bam files were filtered for alignments that overlap the start and end coordinates of the protospacer region of the Cas12a crRNA using bamtools filter -region (bamtools v2.5.1 (ref. 121)). Filtered bam files were loaded into the Integrative Genomics Viewer 2.17.0 (ref. 122) for visualization of individual read alignments. pysamstats fasta type variation (pysamstats v1.1.2) was used to extract per base total read coverage and deletion counts. The fraction of aligned reads harboring a deletion at each base was plotted using custom scripts in R.

Approximately 200,000 to 1 million cells were harvested, resuspended in 300l RNA Lysis Buffer (Zymo, R1060), and stored at 70C until further processing for RNA isolation using the Quick-RNA Miniprep Kit (Zymo, R1055). 3 RNA-seq was batch processed together with samples unrelated to this study using a QuantSeq-Pool Sample-Barcoded 3 mRNA-Seq Library Prep Kit for Illumina (Lexogen cat#139) in accordance with the manufacturers instructions. 10ng of each purified input RNA was used for first-strand cDNA synthesis with an oligo(dT) primer containing a sample barcode and a unique molecular identifier. Subsequently, barcoded samples were pooled and used for second strand synthesis and library amplification. Amplified libraries were sequenced on an Illumina HiSeq4000 with 100-bp paired-end reads. The QuantSeq-Pool data was demultiplexed and preprocessed using an implementation of pipeline originally provided by Lexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). The final outputs of this step are gene level counts for all samples (including samples from multiple projects multiplexed together). Downstream analyses were performed using DESeq2 (ref. 123) for differential expression analysis, crisprVerse124 for off-target analysis, and custom R scripts for plotting as detailed in Supplementary Information.

For the CRISPRi experiments targeting the HBG1/HBG2 TSSs or HS2 enhancer, K562 cells engineered (by lentiviral transduction at MOI~5) for constitutive expression of multiAsCas12a-KRAB were transduced with crRNAs and sorted, followed by resuspension of ~200,000 to 1 million cells in 300l RNA Lysis Buffer from the Quick-RNA Miniprep Kit (Zymo, R1055) and stored in 70C. RNA isolation was performed following the kits protocols, including on-column DNase I digestion. 500ng RNA was used as input for cDNA synthesis primed by random hexamers using the RevertAid RT Reverse Transcription Kit (Thermo Fisher, K1691), as per manufacturers instructions. cDNA was diluted 1:4 with water and 2l used as template for qPCR using 250nM primers using the SsoFast EvaGreen Supermix (BioRad, 1725200) on an Applied Biosystems ViiA 7 Real Time PCR System. Data was analyzed using the ddCT method, normalized to GAPDH and no crRNA sample as reference. qPCR primer sequences are in Supplementary Table 2.

For co-transfection experiments, the day before transfection, 100,000 HEK293T cells were seeded into wells of a 24-well plate. The following day, we transiently transfected 0.6g of each protein construct and 0.3g gRNA construct per well (in duplicate) in Mirus TransIT-LT1 (MIR 2304) transfection reagent according to manufacturers instructions. Mixtures were incubated at room temperature for 30min and then added in dropwise fashion into each well. 24h after transfection, cells were replenished with fresh media. 48h after transfection, BFP and GFP-positive cells (indicative of successful delivery of protein and crRNA constructs) were sorted on a BD FACSAria Fusion and carried out for subsequent flow cytometry experiments.

Approximately 400,000 cells per sample were washed with 1ml cold PBS and resuspended in 400l Pierce RIPA Buffer supplemented with Halt Protease and Phosphatase inhibitor cocktail (Thermo Fisher, 1861281) on ice. Samples were rotated for 15min at 4C, followed by centrifugation at 20,000g for 15min to pellet cell debris. The supernatant was collected and mixed with 4x Bolt LSD Sample Buffer (Thermo Fisher, B0007) supplemented with 50mM DTT, followed by heating for 10min at 70C. Samples were electrophoresed on Bolt 4%12% Bis-Tris Plus Gels (Thermo Fisher), and transferred using the BioRad TurboTransfer system onto Trans-Blot Turbo Mini 0.2m Nitrocellulose Transfer Packs (1704158). Membranes were blocked with 6% BSA in TBST (Tris-buffered saline, 0.1% Tween 20) at room temperature for ~1h, followed by incubation at 4C overnight with antibodies against anti-HA-tag rabbit antibody (Cell Signaling Technology, 3724S) at 1:1,000 dilution and anti-GAPDH rabbit antibody (Cell Signaling Technology, 2118) at 1:3,000 dilution in 6% BSA in TBST. Membranes were washed with TBST at room temperature three times for 5min. each, followed by incubation with IRDye secondary antibody for 1h at room temperature, washed three times with TBST 5min for each and two times with PBS. Blots were imaged using Odyssey CLx (LI-COR).

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-Naimish Patel, M.D., appointed to Chief Medical Officer-

-Julianne Bruno, M.B.A., promoted to Chief Operating Officer-

ZUG, Switzerland and BOSTON, May 23, 2024 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(Nasdaq: CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced the appointment of Naimish Patel, M.D., as Chief Medical Officer, effective May 28, 2024. Dr. Patel is an experienced drug developer who has worked across a wide range of disease areas, including his most recent leadership role as the Global Development Therapeutic Area Head of Immunology and Inflammation at Sanofi. In addition, the Company also announced the promotion of Julianne Bruno, M.B.A., to Chief Operating Officer, effective as of May 23, 2024. She currently serves as the Companys Senior Vice President and Head of Programs & Portfolio Management.

Im thrilled to welcome a transformational leader of Naimishs caliber to the executive team at CRISPR Therapeutics, said Samarth Kulkarni, Ph.D., Chief Executive Officer and Chairman of CRISPR Therapeutics. His extensive drug development experience and proven leadership will be critical as we expand our portfolio and advance multiple assets in our pipeline.

Dr. Kulkarni added: Additionally, I am very pleased to announce Julie's promotion and I look forward to her continued contributions as we scale the Company. Since joining CRISPR Therapeutics in 2019, Julie has been a valuable member of the leadership team and has led several important and impactful cross-functional initiatives including our collaboration with Vertex. With this strengthened executive team, combined with our significant progress to date, CRISPR Therapeutics remains well positioned to rapidly advance our programs and deliver on our mission to develop transformative medicines for patients suffering from serious diseases.

CRISPR Therapeutics' compelling and innovative platform, exciting clinical assets and impressive manufacturing capabilities position the Company to potentially bring several transformative therapies to patients with significant unmet medical need, said Naimish Patel, M.D., I am incredibly excited to join the CRISPR leadership team and help bring these therapies to patients in need.

Dr. Patel joins CRISPR Therapeutics from Sanofi, where he most recently served as the Global Development Therapeutic Area Head of Immunology and Inflammation. Previously, he was the Global Program Head for Dupilumab at Sanofi, leading multiple waves of indication expansion including chronic obstructive pulmonary disease and eosinophilic esophagitis. During his time at Sanofi, Dr. Patel led the development of an industry-leading pipeline across key therapeutic areas including respiratory, dermatology, gastroenterology, and rheumatology. He also oversaw key business development and M&A activities during a rapid phase of pipeline expansion. Dr. Patel is a pulmonary and critical care physician with an extensive background in translational medicine and clinical trials.

Dr. Patel received a B.S. in Mechanical Engineering from MIT and an M.D. from McGill University. He completed his internal medicine training at Columbia-Presbyterian Hospital and his fellowship training in Pulmonary and Critical Medicine at Harvard Medical School. After completing his fellowship, Dr. Patel was a member of the faculty at Harvard and Beth Israel Deaconess Medical Center where he led an NIH-funded lab in translational immunology focused on innate defense functions of the lungs. He previously held positions in clinical development and discovery project leadership at AstraZeneca and Vertex Pharmaceuticals.

Julianne Bruno, M.B.A., has served as Senior Vice President and Head of Programs & Portfolio Management at CRISPR Therapeutics since March 2023. During her time at CRISPR Therapeutics since joining the Company in April 2019, she has taken on positions of increasing responsibility, including leading the hemoglobinopathies partnership with Vertex through the early clinical stage through approval. In addition, she has been responsible for program leadership of our immuno-oncology assets and the program management function across our franchises. Prior to joining CRISPR Therapeutics, Ms. Bruno worked at McKinsey & Company from August 2015 to March 2019 where she was a leader in the biotech practice and served a number of biotechnology companies on a wide range of commercial topics. She received her M.B.A. from The Wharton School and also holds an A.B. from Princeton University.

AboutCRISPR Therapeutics Since its inception over a decade ago, CRISPR Therapeutics has transformed from a research-stage company advancing programs in the field of gene editing, to a company that recently celebrated the historic approval of the first-ever CRISPR-based therapy and has a diverse portfolio of product candidates across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine, cardiovascular, autoimmune and rare diseases. CRISPR Therapeutics advanced the first-ever CRISPR/Cas9 gene-edited therapy into the clinic in 2018 to investigate the treatment of sickle cell disease or transfusion-dependent beta thalassemia, and beginning in late 2023, CASGEVY (exagamglogene autotemcel) was approved in some countries to treat eligible patients with either of those conditions. The Nobel Prize-winning CRISPR science has revolutionized biomedical research and represents a powerful, clinically validated approach with the potential to create a new class of potentially transformative medicines. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer and Vertex Pharmaceuticals. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Boston, Massachusetts and San Francisco, California, and business offices in London, United Kingdom. To learn more, visit http://www.crisprtx.com.

CRISPR THERAPEUTICS standard character mark and design logo are trademarks and registered trademarks ofCRISPR Therapeutics AG.The CASGEVY word mark and design are trademarks of Vertex Pharmaceuticals Incorporated. All other trademarks and registered trademarks are the property of their respective owners.

CRISPR Therapeutics Forward-Looking StatementThis press release may contain a number of forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements made by Drs. Kulkarni and Patel in this press release, as well as statements regarding CRISPR Therapeutics expectations about any or all of the following: (i) its plans for and its preclinical studies, clinical trials and pipeline products and programs, including, without limitation, manufacturing capabilities, status of such studies and trials, potential expansion into new indications and expectations regarding data generally; (ii) the data that will be generated by ongoing and planned clinical trials, and the ability to use that data for the design and initiation of further clinical trials; (iii) the sufficiency of its cash resources; (iv) the expected benefits of its collaborations; and (v) the therapeutic value, development, and commercial potential of CRISPR/Cas9 gene editing technologies and therapies. Without limiting the foregoing, the words believes, anticipates, plans, expects and similar expressions are intended to identify forward-looking statements. You are cautioned that forward-looking statements are inherently uncertain. Although CRISPR Therapeutics believes that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: the efficacy and safety results from ongoing clinical trials will not continue or be repeated in ongoing or planned clinical trials or may not support regulatory submissions; clinical trial results may not be favorable; one or more of its product candidate programs will not proceed as planned for technical, scientific or commercial reasons; future competitive or other market factors may adversely affect the commercial potential for its product candidates; initiation and completion of preclinical studies for its product candidates is uncertain and results from such studies may not be predictive of future results of future studies or clinical trials; regulatory approvals to conduct trials or to market products are uncertain; uncertainties inherent in the operation of a manufacturing facility; it may not realize the potential benefits of its collaborations;uncertainties regarding the intellectual property protection for its technology and intellectual property belonging to third parties, and the outcome of proceedings (such as an interference, an opposition or a similar proceeding) involving all or any portion of such intellectual property; and those risks and uncertainties described under the heading "Risk Factors" in CRISPR Therapeutics most recent annual report on Form 10-K, quarterly report on Form 10-Q and in any other subsequent filings made by CRISPR Therapeutics with the U.S. Securities and Exchange Commission, which are available on the SEC's website at http://www.sec.gov. Existing and prospective investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date they are made. CRISPR Therapeutics disclaims any obligation or undertaking to update or revise any forward-looking statements contained in this press release, other than to the extent required by law.

Investor Contact: Susan Kim +1-617-307-7503 susan.kim@crisprtx.com

Media Contact: Rachel Eides +1-617-315-4493 rachel.eides@crisprtx.com

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CRISPR Therapeutics Strengthens Executive Leadership Team with Key Appointments - GlobeNewswire

CRISPR Therapeutics AG (NASDAQ:CRSP Get Free Report) shares traded down 3.1% during trading on Thursday after Citigroup lowered their price target on the stock from $89.00 to $84.00. Citigroup currently has a buy rating on the stock. CRISPR Therapeutics traded as low as $54.32 and last traded at $55.06. 521,944 shares traded hands during mid-day trading, a decline of 70% from the average session volume of 1,717,410 shares. The stock had previously closed at $56.80.

Several other analysts have also recently weighed in on the company. Needham & Company LLC dropped their price objective on CRISPR Therapeutics from $90.00 to $88.00 and set a buy rating for the company in a report on Thursday, May 9th. Chardan Capital increased their price objective on shares of CRISPR Therapeutics from $110.00 to $112.00 and gave the company a buy rating in a report on Thursday, February 22nd. Wells Fargo & Company lowered their target price on shares of CRISPR Therapeutics from $70.00 to $65.00 and set an equal weight rating for the company in a report on Thursday, May 9th. TheStreet raised CRISPR Therapeutics from a d+ rating to a c rating in a research note on Friday, February 23rd. Finally, Oppenheimer decreased their price objective on CRISPR Therapeutics from $102.00 to $95.00 and set an outperform rating for the company in a research note on Friday, May 10th. Three analysts have rated the stock with a sell rating, seven have assigned a hold rating and eight have issued a buy rating to the company. According to data from MarketBeat, the stock presently has a consensus rating of Hold and a consensus target price of $73.57.

Check Out Our Latest Report on CRSP

Hedge funds have recently modified their holdings of the company. Teacher Retirement System of Texas boosted its position in CRISPR Therapeutics by 5.0% during the 3rd quarter. Teacher Retirement System of Texas now owns 21,284 shares of the companys stock worth $966,000 after acquiring an additional 1,015 shares during the last quarter. California Public Employees Retirement System grew its position in CRISPR Therapeutics by 5.9% in the third quarter. California Public Employees Retirement System now owns 112,169 shares of the companys stock valued at $5,091,000 after purchasing an additional 6,207 shares in the last quarter. Private Advisor Group LLC increased its stake in CRISPR Therapeutics by 12.3% in the 3rd quarter. Private Advisor Group LLC now owns 5,654 shares of the companys stock valued at $254,000 after buying an additional 618 shares during the last quarter. SteelPeak Wealth LLC lifted its position in CRISPR Therapeutics by 14.6% during the 3rd quarter. SteelPeak Wealth LLC now owns 5,780 shares of the companys stock worth $262,000 after buying an additional 735 shares in the last quarter. Finally, Loring Wolcott & Coolidge Fiduciary Advisors LLP MA boosted its stake in shares of CRISPR Therapeutics by 137.9% during the 3rd quarter. Loring Wolcott & Coolidge Fiduciary Advisors LLP MA now owns 1,035 shares of the companys stock worth $48,000 after buying an additional 600 shares during the last quarter. 69.20% of the stock is currently owned by institutional investors.

The company has a market cap of $4.73 billion, a P/E ratio of -20.49 and a beta of 1.80. The company has a fifty day moving average price of $60.59 and a two-hundred day moving average price of $65.51.

CRISPR Therapeutics (NASDAQ:CRSP Get Free Report) last posted its quarterly earnings results on Wednesday, May 8th. The company reported ($1.43) earnings per share for the quarter, missing analysts consensus estimates of ($1.35) by ($0.08). The business had revenue of $0.50 million during the quarter, compared to analysts expectations of $25.53 million. During the same period last year, the company posted ($0.67) EPS. The firms quarterly revenue was down 99.5% on a year-over-year basis. Research analysts anticipate that CRISPR Therapeutics AG will post -5.64 EPS for the current year.

(Get Free Report)

CRISPR Therapeutics is a gene-editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene-editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases.

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CRISPR Therapeutics (NASDAQ:CRSP) Trading Down 3.1% on Analyst Downgrade - Defense World

A healthcare organization that operates clinics inside Seattle public schools says it can now offer gender-affirming hormone treatment to children, emails from a health center program manager reveal.

Emails obtained by The Daily Wire reveal that Neighborcare Health, a healthcare organization that runs clinics in middle and elementary schools in the Seattle Public Schools district, announced in January 2023 that hormone treatments were now included in the gender-affirming care it could offer.

The Neighborcare Health team wanted to share an exciting new update to our School-based Health Center services, the email from a Program Manager for Neighborcare Health, which was obtained by Parents Defending Education through a public records request, reads. Our program will begin offering comprehensive, evidence-based, gender-affirming care services to our students and families who need them.

The change meant that the group could now give hormone treatments to children, it explained. While our School-based Health Centers have long offered a continuum of gender-affirming care services including supportive student counseling, promotion of social identity, and health education, this step will allow us to begin offering families access to important medical interventions including hormone treatments, it states.

The offerings mark one of the boldest efforts to create what Manhattan Institute Fellow Leor Sapir has dubbed the school-to-clinic pipeline whereby young school children are encouraged to undergo irreversible medical interventions that seek to change their sex.

Neighborcare, which runs 14 different school-based clinics in Seattle Public Schools and Vashon Island School District, states that its mission is tohelp students do better in school by working to address health concerns, prevent serious illness, and promote healthy lifestyles. The clinic does not, however, openly admit to providing so-called gender-affirming care, which is conspicuously absent from its services offered list despite announcing that it would begin offeringhormone therapies at some of its school-based clinics over a year ago.

The company official stated in the email that its foray into hormone treatment for children would initially be to a pilot program and that the administration of the irreversible drugs would only occur with parental approval.

These services will be a small pilot available to students and families within the existing schools we serve, the email went on to state, also noting that hormone therapy will only be administered to those who have the approval of parents or legal guardians.

The company defended its decision to offer cross-sex hormone therapy to children in a comment to The Daily Wire, claiming that it was a form of life-saving care.

Gender-affirming care in its many forms represents life-saving care for many of the youth and their families who choose it, spokeswoman Mary Schilder told The Daily Wire. Neighborcare Health offers care for transgender and non-binary students that align with best practices defined by regional and national pediatric care authorities and is aimed at meeting needs expressed by the students and families we serve.

Rhyen Staley, a researcher for Parents Defending Education, which obtained the documents through a public records request, called the administration of irreversible treatments to children in schools unconscionable.

It is deeply concerning that the institutions Americans have traditionally trusted with keeping children healthy and safe are now giving them access to harmful medical practices, Rhyen Staley, a researcher for Parents Defending Education, told The Daily Wire. It is unconscionable that this is happening within the K-12 school setting.

A separate email from an unidentified individual working in public health for Seattle and King County flagged the Neighborcare Health email to people associated with the Seattle Childrens Hospital and Seattle Schools.

I know many of you are already providing different levels of care and support for your students around gender and I simply wanted to share this, the email states, before pointing out that there is the possibility of media interest in the program.

Though medical institutions that turn a profit by administering irreversible cross-sex hormones to children claim that transgender medical interventions are life-saving, European countries to the left of the United States have recently restricted the use of puberty blockers and hormone therapy.Sweden and England have both taken steps to limit the availability of medical interventions that seek to change the sex of patients. The Scandinavian country stopped hormone therapy for minors with few exceptions while Englands National Health Service stopped prescribing puberty blockers.

Some of the most vocal advocates in favor of administering puberty blockers and cross-sex hormones to children actually receive funding from the very same pharmaceutical companies that manufacture the drugs that are given to children suffering from gender dysphoria, Daily Wire investigations have revealed.

Seattle Public Schools, which did not respond to a request for comment, has pushed medical gender transitions more blatantly than perhaps any other school district, but it is far from the only one to encourage children to undergo irreversible medical interventions intended to modify ones sex.

The Daily Wire previously revealed that the Los Angeles Unified School District reached out to a transgender youth clinic at the Childrens Hospital of Los Angeles after staff found out that a student was considering going on testosterone therapy. The district, which frequently pushes transgenderism on students through its lesson plans, book readings, and spirit day celebrations, had just recently hosted members of the Childrens Hospital of Los Angeles to advertise the transgender clinics offerings to parents.

Conservative journalist and activist Chris Rufo revealed that public schools in Chicago teamed up with the Lurie Childrens Hospital of Chicago to promote pro-transgender curriculum and gender-affirming books aimed at children.

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EXCLUSIVE: Health Centers At Seattle Public Schools Offer 'Gender-Affirming' Hormone Therapy To Children - The Daily Wire

DeTar Healthcare System is hosting a hormone replacement therapy information presentation for the Crossroads, targeting women going through menopause or looking to be informed about treatment.

The presentation will be held 6-7 p.m. May 30 in DeTar Hospitals North Education Classroom at 101 Medical Drive and will be presented by gynecologist and obstetrician Dr. Philip Suarez.

HRT is most commonly used as both a menopausal and postmenopausal hormone therapy to treat symptoms associated with female menopause, according to a news release. It can be beneficial to women seeking relief from side effects from menopause, such as vaginal dryness or hot flashes.

Menopause occurs 12 months after a womans last period. The years leading up to that point, when women may have changes in their monthly cycles, hot flashes, or other symptoms, are called the menopausal transition or perimenopause, according to the National Institute on Aging.

The menopausal transition most often begins between ages 45 and 55 but can happen earlier, according to the institute. It usually lasts about seven years but can be as long as 14 years. The duration can depend on lifestyle factors such as smoking, age it begins, race and ethnicity.

Age, family medical history, personal medical history and severity of ones symptoms are important factors to consider when deciding whether or not to take HRT, according to the release.

Were very excited to have Dr. Suarez share his knowledge of HRT, positive effects of taking HRT and the different forms in which its available. We encourage anyone interested in learning more about HRT to attend the presentation, so that they can develop a deeper understanding of their hormone functions before, during and after menopause. said Mary Claire Bradshaw, DeTar Women & Childrens Center director. In addition to the information shared, women will have the opportunity to take note of questions they may have for their primary care provider.

For further questions regarding information or to RSVP, please contact 361-788-6125 or 361-788-6111. Attendees are encouraged to bring a guest to the free presentation.

Link:
DeTar Healthcare System to host hormone replacement therapy seminar - Victoria Advocate

Email communications recently provided to The Daily Wire show that Seattle, Washington schoolchildren can acquire transgender hormone therapy from public school clinics.

[RELATED: Study Shows The Vast Majority of Children Confused About Their Gender Grow Out of The Feelings Without Intervention]

The healthcare organization offering the treatments, Neighborcare Health, operates 14 public school based clinics in middle and elementary schools in Seattle and in the nearby Vashon Island School District.

This clinics are similar in form and function to the school-based clinics at a number of Maine public school districts, including one at Lawrence High School that made headlines last year after her father alleged that they sent his minor daughter home with an unlabeled baggy of Prozac.

[RELATED: Maine Dad Says High School Clinic Sent 17-Year-Old Daughter Home with Secret Baggy of Zoloft, Sicced Child Protective Services on Him For Complaining]

Our program will begin offering comprehensive, evidence-based, gender-affirming care services to our students and families who need them, said a Neighborcare Health program manager in emails originally acquired by Parents Defending Education.

The emails show that hormone therapy has been available in the school clinics since January 2023, but it is unclear how many children have received the drugs.

According to Neighborcare Health, the organization had been providing some gender-affirming care such as counselling, and helping children socially transition, even before they began hormone therapies.

The group celebrated its new ability to provide hormone therapy along with unspecified other important medical interventions.

Although it is unclear what other medical interventions the group can provide, it could possibly refer to puberty blockers, which do irreversible damage to children, and have been restricted in multiple European countries due to their negative effects.

[RELATED: UK Bans Puberty Blockers for Minors as Maine Pushes Sanctuary Status for Gender-Affirming Treatments]

Given that the Neighborcare clinics serve elementary and middle schools, the vast majority of children receiving the hormone treatment and other transgender medical intervention from them would be under 13.

According to the healthcare groups website, the clinics will also offer referrals to gender clinics which will provide further, surgical transgender procedures.

In the emails provided to the Daily Wire, the company claimed that its new hormone treatments are only a pilot program, and will only be prescribed to children with parental consent.

These services will be a small pilot available to students and families within the existing schools we serve, said the company.

It is unclear whether the program is still in its pilot stage, as the emails come from early 2023.

As American liberal organizations and states, including Maine, have pushed forward with radical gender ideology, multiple European countries, including Britain, Norway, and France, have moved to restrict gender transitions for children.

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Students in Seattle Schools can Acquire Hormone Therapy from In-House Clinics: Daily Wire - The Maine Wire

Silverback gorillas are famous for their impressive, bulging physiques and their rather modest genitalia. Now, scientists have uncovered a potential genetic link between these apes' small members and infertility problems in male humans.

Coming in at just 1.1 inches (3 centimeters) long, on average, the penis of the adult male gorilla (Gorilla) is the smallest phallus of all apes. The gorilla's genital size comes with other deficits in its reproductive capacity, such as low sperm count compared to other primates, and sperm with poor motility and a diminished ability to bind to eggs.

Given that these are reproductive issues that can also affect humans, it may seem surprising that all male gorillas share these traits. However, this can be explained by gorillas' mating system, said Jacob Bowman, lead author of the new study and a postdoctoral researcher at the University at Buffalo.

Gorillas operate in a polygynous system, in which a dominant male has near-exclusive access to females in his troop. The silverback's unwieldy physique means it has no problem securing mates, and thus, its sperm doesn't have to compete with that of other males and it can produce offspring without many, highly motile swimmers. The theory is that this lack of sperm competition led to the evolution of gorillas' small genitalia.

Related: Move over, Viagra this spider's boner-inducing venom could treat people let down by the blue pill

This got researchers "wondering if, at a genetic level, we can find genes associated with spermatogenesis [sperm production] or that we see leading to poor-quality sperm," Bowman told Live Science. Gorillas and humans share the vast majority of the same genes so if the researchers could pinpoint suspect genes in gorillas, they could next turn their attention to the human genome.

Roughly 15% of U.S. couples have trouble conceiving, according to Yale Medicine, and more than half of those cases involve male infertility. Around 30% of infertility cases have a genetic basis, said Vincent Straub, a doctoral student in population health at the University of Oxford who was not involved in the new study. However, the genes involved in male infertility are poorly understood.

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To help unravel those genetics, Bowman and colleagues combed through a database of more than 13,000 genes across 261 mammals. This involved looking at genes' underlying sequences, to see how they changed over time in related animals. The aim was to see if certain genes in the gorilla branch of the tree of life were evolving at dramatically reduced rates, Bowman said.

This can happen when there isn't strong pressure to get rid of genetic mutations that could hinder a population's survival such as those related to gorillas' low-quality sperm. This process, called "relaxed purifying selection," can result in seemingly harmful mutations becoming common in a species.

The data turned up 578 genes in the gorilla lineage that underwent this type of selection. An analysis and existing data suggested that many of these genes are involved in making sperm. However, not all the flagged genes had known roles in male fertility.

To better understand these genes' functions, the team turned to the fruit fly (Drosophila melanogaster), a commonly used genetic model in biology. They systematically silenced each of the genes in male flies to see if they affected the insects' ability to reproduce. In this way, they uncovered 41 new genes that hadn't previously been tied to male fertility.

The researchers then connected the dots back to humans using a genetic database with data from 2,100 men with infertility, who either had very low amounts or a lack of sperm in their semen. They also looked at data from fertile men, focusing on the genes they'd flagged in gorillas. They found that, in 109 of relaxed gorilla genes, the infertile men carried more loss-of-function mutations than did fertile men; loss-of-function mutations reduce a gene's ability to make the protein it codes for.

While it's likely these genes are involved in human male fertility, more research is needed to learn exactly how they work in the body. Straub emphasized that infertility is very complex, and that not all of it comes down to genetics. To fully understand it, scientists need to account for how different genes interact with one another and with an organism's environment and its behavior.

The findings drawn from gorillas open the door to future explorations about how these genes, and others closely associated with them, might influence fertility in people, Straub said. The study was published May 9 in the journal eLife.

Ever wonder why some people build muscle more easily than others or why freckles come out in the sun? Send us your questions about how the human body works to community@livescience.com with the subject line "Health Desk Q," and you may see your question answered on the website!

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The same genetic mutations behind gorillas' small penises may hinder fertility in men - Livescience.com

In May 2023, former health minister Lord James OShaughnessy acknowledged in a report that there were many issues with the UKs clinical trial industry. The report showed that the country fell from fourth to tenth globally for trial initiation, with a big drop in the number of Phase III trials initiated.

At the same time, the UKs National Health Service (NHS) has been under extreme pressure with staffing woes, including the recent junior doctor strikes and financial difficulties impeding clinicians ability to participate in research, especially in more demanding cell and gene therapy trials.

With a snap general election now looming on 4 July and the NHS being one of the top concerns for voters, it is more important than ever for political parties to set out their manifesto including how they are going to facilitate research and support the NHS.

The UK Conservative government has tried to improve the countrys ability to develop cell and gene therapies, with a $10m grant in March 2023 for the NHS Blood and Transplant (NHSBT) to open a facility to develop and manufacture new gene and cell therapies called the Clinical Biotechnology Centre (CBC).

Funds have also been provided to several companies through the Life Sciences Innovative Manufacturing Fund (LSIMF) grants, including 151m for Pharmaron and 14m for Touchlight for cell and gene therapy development and manufacturing.

On top of all this, the UKs National Institute for Health and Care Research (NIHR) has announced a 17.9m investment in the Advanced Therapy Treatment Centre Network (ATTC Network).

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A year on from Lord OShaughnessys report and despite the governments investment in the industry, the UK is still struggling to contribute to cell and gene therapy trials says haematologist and chief medical officer of stem cell charity Anthony Nolan Dr Robert Danby. Danby speaks exclusively to the Clinical Trials Arena about the difficulties facing the NHS in contributing to cell and gene therapy research.

Robert Danby (RD): The UK Government has committed to implementing all of Lord OShaughnessys recommendations, and we have seen some progress in the year since. Average trial approval and set-up times have been reduced. The number of people taking part in industry clinical studies is rising. According to the Department of Health and Social Care, 82% of commercial studies are on track.

Despite this, there is still more work to do. A thriving clinical research environment depends on an NHS that has the resources and workforce to support it with trials relying on the UKs network of clinicians, nurses, allied health professionals, statisticians, data managers, and more.

But as organisations such as Cancer Research UK have pointed out, were still a long way from NHS staff having the capacity to prioritise research. A survey of health care professionals revealed nearly four in five clinical researchers described lack of capacity as a substantial or extreme barrier to their work. It means clinical research is at risk of being seen as a nice-to-have, rather than an essential mechanism for bringing new and potentially life-saving therapies to the UK.

The OShaughnessy review said less about tackling the barriers patients face to getting onto a clinical trial. We need to think more ambitiously about the diversity of trial participants, to ensure no groups miss out on the potential improvements to care they offer. We also need to see investment in the real-world data landscape that can play such a pivotal role in advanced cell and gene therapies research.

RD: While we have seen commercial trial capacity increase in the last year, cell and gene therapies present additional research challenges that require a concerted effort to overcome. As a haematologist and in my work at Anthony Nolan, I see this close up.

Blood cancers and other haematological disorders are at the forefront of the oncoming wave of new cell and gene therapies. With over 50 years experience in cell therapies, both allogeneic and autologous haematopoietic cell transplantation, haematology is one of the few medical fields with the necessary skills and expertise to deliver clinical research into these potentially transformative treatments.

But the UKs haematology departments are not yet equipped, or resourced, to deliver this next wave. For example, cell and gene therapies are reliant on apheresis technology to collect the cells which form the basis of new engineered treatments. But current infrastructure is struggling to cope with basic clinical needs for standard indications, never mind additional requirements for therapy development.

Challenges to recruit to cell and gene therapy trials and rigorous regulatory pathways also contribute to the strain faced by NHS teams expected to deliver both basic care and clinical research. And as cell and gene therapy trials move into other areas of medicine like solid cancers or autoimmune diseases, there will need to be a massive increase in resources, training, and education across the breadth of the NHS.

In order to sustain the development of future cell and gene therapies, long-term investment into NHS capacity, education and training is essential. A more streamlined journey from research to regulatory approval and implementation, that is appropriate for the specific requirements of cell and gene therapies, is also needed.

RD: Brexit has compounded many of the issues affecting the UKs position in the global clinical research market. In my field of haematology, the well-recorded loss of NHS and academic staff due to Brexit has had major implications on our ability to carry out not only day-to-day care but also clinical research.

Weve also heard that differences in the regulatory framework and bodies between the UK and Europe post-Brexit have made the industry reluctant to start trials in the UK because implementation into routine care use looks too time-consuming and challenging.

RD: Accelerating Clinical Trials (ACT) is an innovative self-sustaining model to facilitate the delivery of clinical trials for blood cancers and blood disorders.

The ACT operational hub provides services to industry-sponsored commercial trials including access to a national network of recruitment sites, clinical research expertise and operational support and reinvests the income into non-profit academic investigator-led trials.

The OShaughnessy review highlighted that in its first 12 months, ACT attracted investment from two international pharmaceutical companies to deliver practice-informing blood cancer trials. With the income, ACT works with two major national trial acceleration networks which have recruited more than 2,500 patients in recent years, to the benefit of both patients and the UK life sciences sector.

Earlier this year, Anthony Nolan announced its 1m investment into the programme.

RD: We recognise we are seeing a revolution in cell and gene therapies, and the opportunity in the UK is huge. Our nationalised health care system, with strong links to academia, and healthcare data mean there is enormous potential for UK patients to be some of the first to benefit from these innovative and potentially curative therapies. But only if we act now to address issues in NHS capacity, invest in world-leading data systems and simplify the pathway to therapy implementation.

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NHS investment critical to drive cell and gene therapy research - Clinical Trials Arena

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DALLAS May 23, 2024 A new study focused on the gene tied to a rare form of autism spectrum disorder (ASD) called FOXP1 syndrome offers hope that gene therapy might be able to help patients with this condition.

Genevieve Konopka, Ph.D., is Professor of Neuroscience and an Investigator in the Peter O'Donnell Jr. Brain Institute at UTSouthwestern. Dr. Konopka is a Jon Heighten Scholar in Autism Research and holds the Townsend Distinguished Chair in Research on Autism Spectrum Disorders.

In a study published in Science Advances, researchers from UT Southwestern Medical Center found that using gene therapy to restore the Foxp1 gene to adult mice from which it had been deleted before birth restored the activity of other genes whose levels are controlled by Foxp1. This intervention also corrected some abnormal behaviors characteristic of mice that lack Foxp1. The findings could shed light on other forms of ASD as well.

The ability to partially remedy brain pathway changes at later developmental stages suggests that gene therapy may be effectively applied in FOXP1 syndrome and actually normalize symptoms, said Genevieve Konopka, Ph.D., who co-led the study with Jay Gibson, Ph.D. Both are Professors of Neuroscience and Investigators in the Peter ODonnell Jr. Brain Institute at UT Southwestern.

Jay Gibson, Ph.D., is Professor of Neuroscience and an Investigator in the Peter O'Donnell Jr. Brain Institute at UTSouthwestern.

About 200 individuals worldwide have FOXP1 syndrome, a genetic condition caused by mutations in the FOXP1 gene that render it nonfunctional. Along with intellectual deficits, developmental delays, and other symptoms, people with this disease also tend to have ASD or exhibit autistic behaviors. But how the loss of FOXP1 contributes to these symptoms has been unclear, Dr. Gibson explained.

A key circuit thats disrupted in FOXP1 syndrome connects regions of the brain called the cortex, thalamus, and striatum. To better understand FOXP1s involvement in this circuit, Drs. Konopkaand Gibson and their colleagues used a technique to delete this gene in mice in two populations of neurons in the striatum, which receives inputs from both the cortex and thalamus through a chemical called glutamate. Glutamate causes these neurons to fire when its taken up at structures called synapses that connect neurons.

In one population of neurons, the deletion altered the functions of thousands of other genes and caused changes in neuronal responses as well as significant differences in behavior; the animals had problems building nests and spent more time on the edges of their enclosures. When the researchers used a genetic technique to reinstate Foxp1, this intervention normalized how neurons responded to glutamate and restored activity in 78 genes, most known to function in neural synapses. It also normalized some behaviors, such as nesting and time spent in enclosures.

Further study of this gene and the thousands of other genes it regulates couldidentify new targets for pharmaceuticals to treat this condition. Because some of these genes have also been implicated in other forms of ASD, continuing to study FOXP1 could lead to a better understanding and potential treatments for ASD in general, Dr. Konopka noted.

Other UTSW researchers who contributed to this study are first author Nitin Khandelwal, Ph.D., Instructor of Neuroscience; Ashwinikumar Kulkarni, Ph.D., Assistant Professor of Neuroscience; and Matthew Harper, M.S., Research Associate.

Dr. Konopka is a Jon Heighten Scholar in Autism Research and holds the Townsend Distinguished Chair in Research on Autism Spectrum Disorders.

This study was funded by grants from the National Institute of Mental Health (MH126481 and MH102603), the National Institute of Neurological Disorders and Stroke (NS126143 and NS115821), the James S. McDonnell Foundation 21st Century Science Initiative in Understanding Human Cognition Scholar Award (220020467), and the Simons Foundation (573689).

About UTSouthwestern Medical Center

UTSouthwestern, one of the nations premier academic medical centers, integrates pioneering biomedical research with exceptional clinical care and education. The institutions faculty members have received six Nobel Prizes and include 25 members of the National Academy of Sciences, 21 members of the National Academy of Medicine, and 13 Howard Hughes Medical Institute Investigators. The full-time faculty of more than 3,100 is responsible for groundbreaking medical advances and is committed to translating science-driven research quickly to new clinical treatments. UTSouthwestern physicians provide care in more than 80 specialties to more than 120,000 hospitalized patients, more than 360,000 emergency room cases, and oversee nearly 5 million outpatient visits a year.

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UTSW study sheds light on rare form of autism - UT Southwestern

Cell and gene therapy research is crucial for biopharma development, with HiFi long-read sequencing significantly enhancing many sequencing applications throughout the process. Even though AAV sequencing is one of the newest applications of genomics, it is also one of the most promising in disease research today. Adeno-associated virus (AAV) ranks among the most actively experimented upon vehicles for gene therapy.1 Gene therapies using AAV and cell therapies like CAR-T hold the potential to cure previously incurable diseases. CAR-T cell cancer treatments, in particular, are showing great promise in combating this devastating illness. In 2023, the FDA approved several new AAV-based therapies and cell-based gene therapies for treating Duchenne muscular dystrophy, severe hemophilia A, and sickle cell disease.2,3,4

The design of AAV vectors has consequences on gene therapy research, which could be the last stand in the fight against these diseases, and maybe many more. Highly accurate long-read sequencing supports the investigation into design, validation, and optimization of potential gene therapies using such viral vectors.

This is the final episode in our six-part myth-busting series. Today, were debunking common misconceptions about PacBio HiFi sequencing in cell and gene therapy research.

PacBio HiFi sequencing is too expensive to use when developing and assessing or optimizing gene therapy product design, efficacy and potential safety.

This statement is misleading.

Vector design plays a crucial role in gene therapy development success. In cell and gene therapy, where safety is paramount and R&D takes notoriously long, unexpected errors can quickly derail years of work, potentially delaying lifesaving therapies for those who need it. Fullyand accuratelycharacterizing your AAV product can mean reducing the risk of extremely costly failures during clinical trials.

Understanding the full extent of on- and off-target editing, vector or construct integration, and insertional mutagenesis are key components of validating AAV product design and ensuring its manufacturability. Using the exceptional accuracy and lengths of HiFi reads means that you can be confident in your product designs and avoid surprises down the line that require you to go back to the drawing board.

For more, read our best practices for gene therapy product characterization using HiFi sequencing with Dr. Claire Aldridge at Form Bio.

PacBio long reads are only good for de novo genome assemblies.

This statement is incomplete.

HiFi reads are good for assembling genomes, its true, but they can do so much more.

Count cell and gene therapy research are among the many applications you can do with HiFi sequencing. Whether its full-length AAV sequencing, gene editing assessments, plasmid or amplicon library screening, or vector integration, the winning combination of >20 kb reads and 99.9% accuracy with HiFi allows you to detect variants or events that short-read sequencing would miss.

You can use HiFi sequencing for every research stage of AAV gene therapy development:

Discover AAV vectors: discover novel capsids with targeted sequencing Optimize AAV vector design: improve designs by observing the frequency of truncations, fragmentation, and other non-full-length anomalies Confirm mRNA transcripts: quantify isoforms with full-length isoform sequencing Study host integration: understand the frequency of these events, to ensure the potential safety and efficacy of your product Ensure quality in AAV production: compare vectors and unresolved genomes and assess vector preps produced by different platforms

Read more about what you can do with highly accurate HiFi reads for AAV sequencing.

And what about gene editing? HiFi sequencing can power your gene editing research by enabling you to:

Sequence beyond your target to fully understand the extent of CRISPR-Cas9 editing Assess indels and other mutations that result from gene insertion at a CRISPR target locus Detect rare off-target mutations Understand the effects of haplotype and SNVs on gene editing in cases of allele-specific Cas9 cleavage Avoid PCR biases and limitations by using amplification-free approaches

Read more about what you can do with highly accurate HiFi reads for gene editing research.

Accuracy isnt that important for characterizing AAV impurities, such as partial genomes.

This statement is false.

Accuracy is imperative in all areas of science, and the stakes are arguably even higher in cell and gene therapy research, where clinical trials, patient outcomes, and life-changing therapeutics are on the line.

With highly accurate HiFi long reads, you can assess your AAV for impurities, like partial genomes, while at the same time getting critical information about sequence identity. PacBio HiFi sequencing unites the advantages of long reads with Sanger-level accuracy. This means you can monitor and improve AAV discovery and manufacturing with a single technology, reducing the risk of costly failures, which can set development timelines back by months or even years.

Missing crucial information about the quality of your vector can impact the effectiveness of your design. Hear how biopharmaceutical researchers at Homology Medicines are using highly accurate HiFi reads to discover novel vectors and improve their vector designs in this on-demand webinar.

Pairing nanopore with short-read sequencing is the easiest way to characterize AAVs from inverted tandem repeat (ITR) to ITR.

This statement is short-sighted.

Neither ONT nor short-reads can fully resolve ITRs and additional bioinformatics alignment work is necessary when using short reads. Why run multiple assays when you can do it better in one? Highly accurate long-read sequencing combines the accuracy and read length that you need in one experiment. Never again throw away reads because accuracy is too low or because they are too short to align. In addition, long-read sequencing with HiFi reads support a broad menu of applications that are critical for cell and gene therapy product design and development, including:

AAV sequencing Amplicon-based construct screening Gene editing and on- and off-target assessment Full-length plasmid sequencing Vector integration RNA sequencing Whole-genome sequencing

AAV is complex and difficult to sequence using PacBio HiFi sequencing.

This statement is incorrect.

PacBio and our partners at Form Bio offer an end-to-end workflow for AAV sequencing and data analysis, in an all-in-one solution to optimize your AAV vector designs. This workflow accommodates both ssAAV and scAAV sequencing using the same protocol. Form Bio workflows are certified PacBio compatible and provide analysis software to help you analyze and visualize your AAV data, so you can save time and resources.

With this protocol, you can use HiFi reads to:

Sequence tissues for novel AAV vector discovery Improve vector design Identify impurities, truncation events, and host integration events

See how HiFi sequencing makes it easy to sequence AAV genome populations to identify truncation, mutation, and host integration events.

As weve shown here and throughout our myth-busting series PacBio HiFi sequencing can benefit almost any genomics application by virtue of its long read lengths and exceptional accuracy. Leave those dated misconceptions in the past and start using the power of HiFi sequencing to fuel tomorrows groundbreaking discoveries.

Did you miss the other installments in our myth-busting series? Dont worry, you can catch up here:

Part 1 HiFi sequencing Part 2 human genomics Part 3 cancer genomics Part 4 plant and animal genomics Part 5 microbiology

Are there any other myths about long-read sequencing that you want busted? Let us know! Speak with a PacBio scientist to find out what you can do with HiFi sequencing.

Learn more

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Long-read sequencing myths: debunked. Part 6 cell & gene therapy - Pacific Biosciences

This years prize carrying a gold medal and a $1.2 million award is shared by Stuart Orkin, MD, a researcher at the Dana-Farber/Boston Childrens Cancer and Blood Disorders Center, and Swee Lay Thein, PhD, senior investigator and chief of the sickle cell branch of the National Heart, Lung, and Blood Institute at the National Institutes of Health.

Their work unveiled the genetic and molecular mechanisms underlying the transition in the production of fetal to adult hemoglobin. These discoveries led to work that culminated in the development and approval of Casgevy (exagamglogene autotemcel), the first gene-editing therapy based on CRISPR/Cas9 to be approved for SCD and transfusion-dependent beta thalassemia, a related blood disorder.

Receiving The Shaw Prize is an honor and a testament to the dedication of countless researchers who have contributed to our understanding of hemoglobin regulation over the years, Orkin, who is also a professor of pediatrics at Harvard Medical School, said in apress release from Boston Childrens.

This recognition underscores the potential of our findings to revolutionize the treatment landscape for sickle cell anemia and [beta] thalassemia, offering new hope to patients worldwide, Orkin added.

Often referred to as the Nobel of the East, the prize recognizes scientists who have made striking contributions to research. It was established in 2002 by Run Run Shaw, a philanthropist. To date, 41 individuals have been named Shaw Prize Laureates in Life Sciences and Medicine. There also is a Shaw Prize in Astronomy, and one in Mathematical Sciences.

This years science winners were recognized for their transformative research (Thein) and elegant work (Orkin), according to a Shaw Prize webpage highlighting their careers.

Over the course of their distinguished careers, Swee Lay Thein and Stuart Orkin each made wide-ranging, independent contributions to the analysis of blood cell disorders. Their work intersected when they made complementary and reinforcing discoveries that led to the development of a therapy to treat sickle cell disease and [beta] thalassemia, the webpage states.

Several versions of hemoglobin, the protein that carries oxygen in red blood cells, can be found in the human body. As its name suggests, fetal hemoglobin is produced while a baby develops in the womb. This version of hemoglobin is replaced after birth by an adult form of the protein that is less effective at transporting oxygen throughout the body.

The production of these different versions of hemoglobin is controlled by different genes. The HBB gene, which contains instructions for making a subunit of adult hemoglobin, is mutated in SCD, resulting in the production of a faulty version of the adult form of the protein. Conversely, the HBG gene, which provides instructions for making a component of fetal hemoglobin, is not affected by SCD-causing mutations.

One of the therapeutic strategies that can be adopted to ease the severity of SCD and beta thalassemia is to reactivate the production of fetal hemoglobin an approach that came on the heels of both Orkins and Theins discoveries.

Theins research led to the identification ofthe BCL11A gene as a regulator of fetal hemoglobin production. Follow-up work by Orkin confirmed the BCL11A protein was involved in the process of shutting off the production of fetal hemoglobin after birth a discovery that has rendered him the recipient of several other awards.

These findings led scientists to postulate that reducing BCL11A levels could boost the production of fetal hemoglobin, which in turn would compensate for the faulty or deficient version of the adult form of the protein in SCD and related blood disorders. Such a mechanism is the rationale behind the gene-editing therapy Casgevy, approved in the U.S. earlier this year.

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Shaw Prize awarded to 2 scientists for work in SCD gene therapies - Sickle Cell Disease News

Scientists at the Broad Institute of MIT and Harvard have engineered a harmless adeno-associated virus (AAV) that can efficiently reach the brain, potentially improving the efficacy of brain-targeted gene therapies for neurological conditions such as Huntingtons disease.

Current AAVs that deliver gene therapies to cells in the body via injection into the bloodstream cannot efficiently cross the blood-brain barrier (BBB), the highly selective membrane that regulates what substances from the bloodstream can access the central nervous system (CNS), which consists of the brain and spinal cord.

In a mouse model modified to produce the human version of an important BBB protein, the newly engineered AAV was more widely distributed across the brain and more effective at delivering a gene therapy to brain cells than AAV serotype 9, which is used in an approved CNS-targeted gene therapy.

The study, An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery, was published in Science.

Since we came to the Broad weve been focused on the mission of enabling gene therapies for the central nervous system, Ben Deverman, PhD, the studys senior author and director of vector engineering at the Broads Stanley Center for Psychiatric Research, said in an institute press release. If this AAV does what we think it will in humans based on our mouse studies, it will be so much more effective than current options.

Ken Chan, PhD, one of the studys first co-authors and group leader in Devermans group, said the AAVs have the potential to change a lot of patients lives.

In Huntingtons, defects in the HTT gene lead to toxic clumps of the huntingtin protein, which are thought to damage various parts of the brain, triggering the onset of symptoms.

Gene therapy, which works by delivering to cells genetic material meant to counterbalance a genetic defect, can potentially treat disorders caused by mutations in a single gene, such as Huntingtons.

Most of these therapies use modified, harmless AAVs to deliver the therapeutic cargo to cells. However, current AAVs cannot efficiently cross the BBB, which protects the brain from harmful substances in the blood while allowing essential nutrients and certain molecules to pass through.

To address these limitations, the researchers engineered an AAV that binds to the transferrin receptor (TfR1), a cell surface protein that is highly present in the human BBB and is an established target of antibody-based therapies designed to reach the brain.

To find such a virus, they first screened large AAV libraries in test tubes. Top candidates were then tested in cells and mice (in vivo) modified to produce human TfR1. Screening against a human protein in mice was done to improve the chances that gene therapies using these AAVs would work in human patients.

Weve learned a lot from in vivo screens but it has been tough finding AAVs that worked this well across species, said research scientist Qin Huang, PhD, the other co-first author of the study. Finding one that works using a human receptor is a big step forward.

Injecting the top AAV candidate, called BI-hTFR1, into the bloodstream of TfR1-modified adult mice dramatically increased levels of the AAV in the CNS compared with unmodified mice. This demonstrated that the AAV was indeed crossing the BBB by binding to human TfR1.

In different brain regions, BI-hTFR1 reached up to 71% of nerve cells and 92% of astrocytes, specialized cells in the brain that provide support to nerve cells.

The team then compared BI-hTFR1 with AAV9, an AAV used as a delivery vehicle for Zolgensma (onasemnogene abeparvovec-xioi), a gene therapy approved for the neuromuscular disorder spinal muscular atrophy.

They found that BI-hTFR1 levels in brain tissue were up to 50 times higher than those of AAV9.

As a model for gene therapy, researchers used the new AAV to deliver a healthy copy of the human GBA1 gene, which encodes an enzyme called beta-glucocerebrosidase, to the modified mice. Mutations in this gene cause Gaucher disease and are linked to Parkinsons disease.Both of those are neurological conditions.

Compared with AAV9, BI-hTFR1 delivered 30 times more copies of the GBA1 gene to brain cells and substantially increased the beta-glucocerebrosidase activity in the brain and cerebrospinal fluid, which surrounds the brain and spinal cord.

When we think about gene therapy for a whole-brain disease you need really systemic delivery and broad biodistribution in order to achieve anything, said Eric Minikel, PhD., senior group leader at the Broad. Naturally occurring AAVs just arent going to get you anywhere, Minikel said. This engineered [AAV] opens up a world of possibilities.

In addition to targeting a human protein, the fact that BI-hTFR1 has a similar production yield to AAV9 using scalable manufacturing methods makes it ideal for CNS-targeting gene therapy, the researchers noted.

Researchers at Apertura Gene Therapy, a biotech company co-founded by Deverman, are already developing new CNS-targeting gene therapies using the new AAV.

The scientists believe that further research can help improve the AAVs gene-delivery efficiency to the CNS, the institute said. It may also help reduce AAV accumulation in the liver, and prevent their inactivation by antibodies in some patients two known complications of current viral-based gene therapies.

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New viral carrier shows promise for brain-targeted gene therapies - Huntington's Disease News

The traditional randomised controlled trial (RCT) model has been used for decades in drug development. Although it is considered the gold standard of trial designs, sponsors are increasingly using alternative trial designs, especially those for developing cell and gene therapies.

The unique nature of cell and gene therapies, how they are administered, the complex dosing schedule, and the specialised patient population can make it difficult to use a traditional RCT model.

Innovative trial designs, like single group assignment, adaptive, basket, umbrella and platform trials, allow flexibility to be built into a trial, which experts describe as crucial in running cell and gene therapy trials.

Chief medical and scientific officer at UK stem cell charity Anthony Nolan, Dr. Robert Danby, said: Prospective RCTs have traditionally been the gold standard to evaluate the efficacy and safety of new therapies. For emerging cell and gene therapies, modern trial techniques like adaptive trials offer a promising alternative. These modern trial designs could offer patients new and better treatments sooner and do this without compromising on the quality of data required for new therapies to be approved.

Despite data showing cell and gene therapy trials continue to be designed as RCTs, there is agreement amongst the industry professionals that RCTs may not be the best model, says Dr. Odelia Chorin, rare disease paediatrician and clinical geneticist at the Safra Childrens Hospital, Sheba Medical Center.

GlobalDatas Pharmaceutical Intelligence Centre shows that the single-group assignment trial design is by far the most used approach for cell and gene therapies.

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The main reason for using alternative trial designs is to ensure trials are providing robust and interpretable data, says Amy Raymond, PhD, Executive Director Therapeutic Strategy Lead, Cellular and Genetic Medicines, at contract research organisation (CRO) Worldwide Clinical Trials.

While single assignment trials have been the leading design in cell and gene therapy trials in the past decade, Erin Griner PhD, associate director in clinical research methodology at Worldwide Clinical Trials says she is mostly seeing adaptive designs being used by sponsors.

The thing that everybody in the process wants is optimal, efficient data, but the question is how do we get there? Raymond asks.

Ultimately, the design is heavily influenced by the specific characteristics and requirements of both the therapy and the patient population rather than the indication itself, explains Neta Shanwetter Levit, Clinical Operations Lead, PhaseV, a company that develops machine learning (ML) technology to optimise clinical trial design and analysis.

We are seeing a gradual increase in the number of adaptive trials and basket trials. Platform trials are not as frequent in this area yet, but are starting to increase, Shanwetter added.

Dr. Beatrice De Vos, chief medical officer for EXO Biologics, a biotech developing exosome-based cell therapies, said that the flexibility to change protocols is the most helpful element of alternative trial designs, especially at early stages.

The data collected in an adaptive trial is impacting the development immediately, impacting your next steps in clinical trial phases. That is different to the classic model, so I am very much in favour of the adaptive design, De Vos added.

There is some differentiation between the choice of trial designs based on the indication. GlobalDatas Pharmaceutical Intelligence Centre shows following single assignment and RCTs are more common in oncology studies than in other indications while adaptive trials are mostly used to study treatments for genetic disorders.

The challenges in rare disease studies are the limited patient population and the scarce data, Shanwetter said. Therefore, adaptive trials are particularly useful in rare diseases due to their flexibility. Basket trials are frequently used in oncology to test therapies targeting specific genetic mutations across different types of cancers.

Griner agrees saying the basket trial design is used more often in oncology trials.

Despite it being difficult to randomise a gene therapy trial, in ophthalmology, sham controls are commonly used, Raymond says. But synthetic controls are also more commonly used in oncology while rare diseases are often studied in open-label trials and biomarkers are used to measure endpoints, she adds. Chorin agrees that given the lack of comparator, the industry needs to use biomarkers to measure clinical change.

Given how many different types cell and gene therapies, the design of a trial should be tailored on a drug-by-drug basis and there isnt a one-size-fits-all solution, says Shanwetter.

Another benefit of alternative designs is they allow sponsors to compare the treatment arm with real-world or natural history data using synthetic arms.

Natural history data is collected from patients who received no intervention and allows investigators to have a baseline to measure the drugs efficacy. Such approaches have been used in notable gene therapy approvals, Raymond said.

Zolgensma [pivotal trials] used natural history data as a control, which saw the PIs coming together and doing that research. That in my opinion is what really enabled this gene therapy trial to succeed was using that baseline comparison, Raymond explained.

Novartis gene therapy Zolgensma (onasemnogene abeparvovec) was approved for treating spinal muscular atrophy (SMA) in May 2019.

The ability to use existing data as a control is particularly helpful in rare disease trials where there are few patients, which often leads to trials being terminated due to low recruitment. Additionally, patients also might be more willing to participate if they know they will receive the study drug, Griner adds.

For some rare diseases, we are seeing 80% to 90% of patients being willing to try a gene therapy that is as yet unproven because it gives them some hope. Having a trial design that is an open-label study really increases interest and as a result recruitment, Griner adds.

De Vos says it is possible to match historical data to a degree that is similar to classical placebo, but acknowledges in statistical terms, it is not a head-to-head comparison. De Vos says EXO Biologics is running a Phase I/II trial (NCT06279741) of using a single group assignment design. The Phase I portion of the trial, which is evaluating EXOB-001, an exosome-based cell therapy for premature babies with bronchopulmonary dysplasia, is using natural history data as a comparator while the Phase II stage will be a standard RCT model.

The only challenge with using natural history data as a control, Griner explains, is that in some diseases there is very little available.

Overall, Griner says that agencies have been supportive of studies with alternative designs, adding that the US Food and Drug Administration (FDA) has also released guidance on confirmatory evidence.

De Vos said that there is also guidance available in Europe but that it should be read very carefully. [However] having gone through all these guidelines, companies should not refrain from coming up with new designs, rather than sticking to the old fashioned ones to extract the most out of their observation in terms of new product development, De Vos concluded.

Raymond says the FDA and EMA are often aligned, but not always, recalling instances where a trial has required region-specific protocols, and in some cases region-specific primary endpoints.

One thing the regulators need to be looking at is having a more unified framework with alternative trial designs. Its not a small ask but it should be a real priority as it would be helpful to the industry as a whole, Raymond concluded.

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Flexibility of alternative trial designs crucial for cell and gene therapy research - Clinical Trials Arena

According to latest study, the global cell & gene therapy bioanalytical testing services market size was estimated at USD 585.19 million in 2023 and is projected to hit around USD 1,194.92 million by 2033, growing at a CAGR of 7.4 % during the forecast period from 2024 to 2033.

The emergence and proven efficacy of novel modalities such as cell and gene therapies have significant growth in the pharmaceutical industry. This growth is derived by substantial investments from drug developers, particularly in areas like rare diseases and immune oncology, where these therapies have demonstrated notable success.

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Cell & Gene Therapy Bioanalytical Testing Services Market Overview

The rapid growth of the cell and gene therapy bioanalytical testing services market is driven by the potential of these therapies to revolutionize treatment across a spectrum of diseases, from cancer to genetic deficiencies. This innovation necessitates new considerations for clinical testing, prompting biopharmaceutical developers to seek quality laboratory testing partners or subject matter experts (SMEs) to navigate the unique challenges of bioanalytical testing in this domain. Such partnerships are essential for driving innovation and ensuring successful development programs as they progress towards regulatory approval.

Gene and cell therapies, utilizing viral or non-viral vectors, present distinct challenges, with gene therapies classified into gene correction, gene editing, and oncolytic virus therapies, while cell therapies encompass adoptive cell and stem cell therapies. Consequently, specialized expertise in CGT solutions and services is crucial, particularly in areas like pharmacokinetics (PK), immunogenicity, and pharmacodynamics (PD) biomarker analysis, where advanced techniques and meticulous care are paramount.

Cell & Gene Therapy Bioanalytical Testing Services Market Key Takeaways

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Cell & Gene Therapy Bioanalytical Testing Services Market Dynamics

Driver

Increasing research and development

The increasing growth of gene and cell therapies in treating previously incurable diseases is the beginning of a transformative era in healthcare. With pharmaceutical companies heavily investing in research and development to unlock the full potential of this field, a surge of new gene and cell therapy products is entering early development stages. Rising trends in research, product releases, and patent applications becomes crucial for identifying global innovation patterns and seizing commercial opportunities. As companies strive to make informed decisions about resource allocation, the demand for specialized cell and gene therapy bioanalytical testing services is escalating. These services play a vital role in ensuring the safety, efficacy, and regulatory compliance of innovative therapies, thus driving the growth of this dynamic market segment.

Restraint

Scalability Challenges

The scalability of cell and gene therapy manufacturing emerges as a pivotal challenge hindering market growth. Manual labor-intensive processes contribute to time and cost inefficiencies, making manufacturing both laborious and expensive. Intricate nature of these therapies, their production remains notoriously costly, thereby limiting accessibility for patients and impeding scalability. The cell and gene therapy bioanalytical testing services market experience constraints as the industry grapples with overcoming these scalability hurdles.

Opportunity

Evolution of Cell and Gene Therapy

The gene editing techniques and transgene delivery systems is revolutionizing patient care, with promising outcomes across various disease domains, including rare diseases and challenging cancers. As the gene and cell therapy field continues to evolve, scientists recognize the need for personalised quantitative measurements specific to their drug programs. This demand for advanced analytics extends beyond traditional methods like chromatography and ligand binding assays. An opportunity arises for the cell and gene therapy bioanalytical testing services market to meet the evolving needs of drug developers by offering innovative solutions and cutting-edge technologies, thereby facilitating further advancements in this burgeoning field.

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By Test Type

The bioavailability & bioequivalence studies dominated the market with a share of 35.22% in 2023, holding a significant share and poised for rapid growth in the forecast period. These studies play a critical role in assessing the in vivo biological equivalence of different proprietary preparations of a drug, ensuring consistency and effectiveness. Bioequivalence studies ascertain whether two pharmaceutical products are essentially interchangeable, while bioavailability studies measure the concentration of a drug in the plasma or blood over time following a systematic protocol. As pharmaceutical companies strive for quality assurance and regulatory compliance, the demand for these essential tests continues to escalate, driving growth in the market segment.

The pharmacokinetic segment is anticipated to experience significant growth throughout the forecast period. Pharmacokinetics, a pivotal field within pharmaceuticals, focuses on understanding how drugs move within the body and how the body interacts with these drugs. Through the application of terms, theories, and equations, practitioners can accurately estimate drug concentrations in various bodily regions. As the importance of pharmacokinetic testing continues to be recognized in drug development and clinical practice, the demand for these services is expected to rise steadily in the coming years.

By Stage of Development Product Type Insights

The non-clinical segment dominated the market with a market share of 66.12% in 2023 and is also expected to witness the fastest growth at a CAGR of 7.5% during the forecast period and poised for rapid growth in the forecast period within the Stage of Development Product Type category. These studies encompass a range of protocols, including animal studies conducted in accordance with Good Laboratory Practice (GLP) regulations. As pharmaceutical companies prioritize safety and efficacy in drug development, the demand for robust non-clinical studies is expected to continue its upward trajectory, driving growth in this market segment.

The clinical segment is poised for growth in the forecast period within the Stage of Development Product Type category. Clinical, comprising three key phases, serve as pivotal stages in the development of new medicines, ensuring their safety, efficacy, and optimal dosage forms before they can be approved for patient use. These are designed to determine the optimal dosage, formulation, safety profile, absorption characteristics, and therapeutic effectiveness of a drug. As pharmaceutical companies strive to bring innovative treatments to market while adhering to rigorous regulatory standards, the demand for clinical trial services is expected to increase, driving growth in this segment.

By Indication Insights

The oncology segment dominated the market and accounted for the largest revenue share of 49.85% in 2023, capturing the largest revenue share within the indication category. Cell and gene therapies represent a revolutionary approach in cancer treatment. These therapies operate by modifying the DNA of a patient's existing cells, providing them with new instructions to detect and combat cancer. By binding the inherent capabilities of the immune system, cell and gene therapies offer a promising avenue for more effective and personalized cancer treatment strategies. As research in this field continues to advance and more therapies gain regulatory approval, the oncology segment is expected to maintain its stronghold in the market.

The rare diseases segment is poised for rapid growth during the forecast period within the indication category. Cell and gene therapies have demonstrated notable efficacy, in addressing clinical indications associated with rare diseases. These therapies hold promise for patients with serious or life-threatening rare diseases by targeting the underlying cause of the condition, rather than merely alleviating symptoms. As research and development efforts continue to expand in this area and more therapies advance through clinical trials, the rare diseases segment is expected to experience significant growth, providing new hope for patients and caregivers alike.

By Product Type Insights

The cell therapy segment dominated the product segment market with a market share of 42.19% in 2023, capturing a significant market share. The potential applications of cell therapies are vast and encompass a wide range of medical conditions, including cancers, autoimmune diseases, urinary problems, infectious diseases, joint cartilage damage, spinal cord injuries, immune system deficiencies, and neurological disorders. Cell therapy encompasses a diverse array of approaches, including both stem cell- and non-stem cell-based therapies, as well as unicellular and multicellular therapies. As research and development in cell therapy continue to advance, fueled by promising clinical outcomes, the market for these innovative treatments is expected to witness sustained growth.

The gene-modified cell therapy segment is poised for the fastest growth during the forecast period within the product category. These therapies, often referred to as Ex Vivo gene therapies, involve modifying cells outside the patient's body and then reintroducing them to combat the disease, as seen in Chimeric antigen receptor T-cell (CAR T-cell) therapy for cancers. Gene-modified cell therapy entails precisely modifying cells ex vivo to enhance the patient's ability to fight the disease, representing a promising frontier in personalized medicine. As advancements in genetic engineering continue to drive innovation in this field, the gene-modified cell therapy market is expected to experience significant growth.

Regional Insights

North America dominated the cell & gene therapy bioanalytical testing services market and accounted for the largest revenue share of 40.56% in 2023, capturing the largest revenue share. Despite the FDA's approval of only a handful of gene therapy treatments, numerous ongoing studies indicate a burgeoning interest in this field. Gene therapy is currently under investigation for a wide array of diseases, including cancer, heart disease, cystic fibrosis, sickle cell disease, and haemophilia A. CAR-T cell therapies stand out as the primary technology utilized in the pipeline of genetically modified cell therapies, representing 52%, followed by a diverse range of technologies grouped under the "other" category at 27%. Notably, 97% of CAR-T cell therapies are focused on cancer indications, with a minority targeting non-oncology areas such as scleroderma, HIV/AIDS, and autoimmune disease. The robust pipeline of cell and gene therapies in the US underscores significant scientific advancements, raising questions about the extent to which these innovations will translate into tangible progress within the US healthcare system.

The Asia Pacific region is poised to experience the fastest CAGR of 7.8% growth in the cell and gene therapy sector during the forecast period. Recent developments in this space across countries like Singapore, China, South Korea, and Japan have been remarkable, with these nations emerging as dynamic hubs for pioneering research. Significant investments from governments and institutions have facilitated the establishment of state-of-the-art facilities and the adoption of cutting-edge technologies. As Asia Pacific continues to make strides in this field, it presents lucrative opportunities for collaboration and innovation in the global healthcare landscape.

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Cell & Gene Therapy Bioanalytical Testing Services Market Recent Developments

Cell & Gene Therapy Bioanalytical Testing Services Market Top Key Companies:

Cell & Gene Therapy Bioanalytical Testing Services Market Report Segmentation

This report forecasts revenue growth at country levels and provides an analysis of the latest industry trends in each of the sub-segments from 2021 to 2033. For this study, Nova one advisor, Inc. has segmented the Cell & Gene Therapy Bioanalytical Testing Services market.

By Test Type

By Product Type

By Stage of Development by Product Type

By Indication

By Region

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Cell & Gene Therapy Bioanalytical Testing Services Market Size to Hit USD 1.19 Bn by 2033 - BioSpace

PHILADELPHIA PA Greater Philadelphia has emerged as a powerhouse in the cell and gene therapy industry, housing almost 10 percent of the worlds companies in this rapidly expanding sector. A report commissioned by the Chamber of Commerce for Greater Philadelphia has revealed that this industry now provides employment to more than 7,000 people in the region.

Leading the Way: Cell and Gene Therapy in Greater Philadelphia, hailed as the most comprehensive regional snapshot of the sector, declares cell and gene therapy as a paramount economic catalyst for the region in the last decade. The area displays a rich blend of established and emerging businesses, leading research institutions, and an exceptional pool of industry talent that has collectively sparked a wave of economic prosperity and groundbreaking treatments for myriad diseases.

Chamber President and CEO, Chellie Cameron, is effusive about the regions role, noting, Greater Philadelphia is the top destination for those who wake with a mission to improve lives through cell and gene therapy treatments. The region offers everything that research companies, researchers, and their support teams would want, which is why we continue to make such great strides.

The report, compiled by economic research firm Ninigret Partners LLC and supported by a grant from the Knight Foundation, will serve as a beacon for community members, talent, policymakers, and practitioners seeking to advance parallel sector-specific growth strategies both locally and internationally.

John Churchill, Director, Philadelphia, Knight Foundation acknowledged the regions pioneering role in his statement: Greater Philadelphia is already a global leader in cell and gene advanced therapies and is poised to become one of the top innovation hubs in this field. This study aims to pinpoint the key factors and resources driving success.

Tracking back to the origins of cell and gene therapy discovery 25 years ago in Philadelphia, Claire Greenwood, Executive Director and Senior Vice President of Economic Competitiveness for the Chamber, observes how this mature ecosystem now includes a broad range of economic drivers. The report reveals Greater Philadelphia to be home to 60 of the estimated 500 cell and gene therapy companies worldwide. In addition, the region claims a top-ranking position for NIH funding in gene therapy and is a leader in translational science.

Furthermore, the region has seen a substantial influx of capital into life sciences companies. Since 2018, 547 life sciences companies in Greater Philadelphia have accumulated $18.7 billion through various financial avenues, with approximately $8 billion specifically related to cell and gene therapy.

In addition to the regions robust lineup of cell and gene therapy companies, the Greater Philadelphia ecosystem also showcases a well-developed supportive infrastructure. Recent additions include a Charles River CRADL facility, the entry of Mispro biotech services, the creation of the Cencora CGT Integration Hub, and the IBX Advanced Therapeutics Network.

The Chambers CEO Council for Growth and 11 partner companies, institutions, and universities launched the Cell & Gene Therapy and Connected Health Initiative in January 2019. The main objectives of this Initiative include shared storytelling, critical infrastructure development, talent assessment and attraction, and ecosystem scalability. Greenwood highlights that this new study will further enhance the innovative and collaborative approaches emanating from the Initiative.

As the Greater Philadelphia region continues to establish its dominance in the cell and gene therapy landscape, its clear that its synergistic blend of economic resources, research capabilities, and industry talent is driving both an economic boom and life-altering therapeutic developments. The implications of this include not only a thriving job market but also the promise of new treatments that could revolutionize healthcare for countless individuals worldwide.

For the latest news on everything happening in Chester County and the surrounding area, be sure to follow MyChesCo on Google News and Microsoft Start.

Originally posted here:
Greater Philadelphia Dominates Cell and Gene Therapy Landscape - A Beacon of Innovation and Employment - MyChesCo

Photo:VCG

The team led by Shao Zhicheng created a revolutionary cryopreservation method, dubbed MEDY, which preserves the structural integrity and functionality of neural cells, allowing for the preservation of various brain tissues and human brain specimens. This advancement holds immense promise not only for research into neurological disorders but also opens up possibilities for the future of human cryopreservation technology.

Professor Joao Pedro Magalhaes from the University of Birmingham K expressed profound astonishment at the development, hailing the technology's ability to prevent cell death and help preserve neural functionality as nothing short of miraculous. He speculated that in the future, terminally ill patients could be cryopreserved, awaiting cures that may emerge, while astronauts could be frozen for interstellar travel, awakening in distant galaxies.

The news has sparked fervent discussions on social media platforms, with many netizens drawing parallels to the concepts depicted in Chinese writer Liu Cixin's science fiction The Three-Body Problem. Interest in the feasibility of future human cryopreservation technology has surged, with individuals expressing a willingness to participate in human trials, eagerly anticipating awakening in a new era within robotic bodies.

"Now we just need a probe that travels at 1-percent speed of light, and can operate for thousands, millions of years on its own power while avoiding space debris, to reach the fleet of ships that's most of the way here already, as Three-Body Problem has illustrated," one netizen posted.

As the boundaries of possibility continue to expand, the realm of cryonics stands on the precipice of a profound transformation, offering glimpses into a future where the line between science fiction and reality grows increasingly blurred. Questions have also emerged as the boundaries expand: Will all the information and memory be indestructibly preserved too? Or, do we really have soul?

Global Times

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Chinese researchers successfully revive human brain frozen for 18 months - Global Times

Words by Melissa Harvey

I might still be 25 in my head, but theres no avoiding the fact Im two decades older when I look in the mirror. In the last couple of years, things have definitely gone south (literally). The fine lines around my eyes are starting to look more like crevices, the wrinkles in my forehead dont soften however well rested I am and my skin seems permanently tired and dull. Though Ive always steered clear of Botox, Id started to wonder if I should bite the bullet and give it a go.

Then I heard about the space age-sounding epigenetic skincare. Epigenetics is the study of the way genes are controlled in the body including how lifestyle and environment can affect the way genes work. Unlike genetic changes, epigenetic changes can be stopped and even reversed and are thought to be responsible for 75% of the ageing process. Needless to say, beauty companies have been researching epigenetics for years in the hope of finally finding the ultimate anti-ageing holy grail.

Based in the US, ABG Lab has now developed the worlds first mesotherapy treatments that use this science to turn the clock back on ageing skin. Its launched four treatments at The Harley Street Skin Clinic in London which target mature skin cells with a range of intradermal injectables.

One (MesoEye C71) is designed to target dark circles and eye bags, another (MesoSculpt C71) breaks down fat cells to sculpt and define, smooth cellulite and tighten skin. A third (Meso-Xanthin F199) is best for polluted, damaged skin and photo-ageing and is aimed at those in their 20s and 30s.

Finally, Meso-Wharton P199 is best for those in their 40s and 50s like me. It uses ABG Labs Whartons Jelly Peptide containing synthetic embryonic peptides to target stem cells deep in the skin to keep them functioning well for as long as possible. Its even been claimed that it can reinvigorate the stem cell activity of someone in their 40s to levels of a 25-year-old which had me racing to book in with Dr Aamer Khan, founder of The Harley Street Skin Clinic.

Meso-Wharton speaks to the stem cells and regenerative cells and gets them to behave like when you were younger, he tells me. As we get into our 50s, were reaching the point where very few of our cells are producing collagen. It stimulates the stem cells to produce more collagen so you get younger looking skin.

The bad news is its likely not for you if you hate needles. Four to six treatments are recommended, around a week or two apart, and each treatment involves wait for it around 200 quick intradermal injections all over the face. Though Im told the needle is much smaller than the one used for Botox, its still a little painful even though a numbing cream is applied first. However, I did find each subsequent treatment hurt less, perhaps because I knew what to expect plus its all over in less than ten minutes.

Downtime takes a lot longer. Though some people look normal within 24 hours, it took my skin around six days to settle after each treatment. For the first couple of days, my entire face was covered with small bumps like mosquito bites and I had some bruising after that, though this could be covered with makeup. This would be significantly less obvious in someone without my pale skin however.

About a month after my final treatment, I start to notice small changes. I still look exactly like myself theres no ironed forehead effect but just a bit better and more refreshed. The shadows under my eyes are less obvious and my wrinkles are softer and shallower. My skin looks and feels more hydrated, particularly first thing in the morning. Suddenly, people start unexpectedly telling me I look well (aka less like an exhausted wreck than usual).

Amazingly, epigenetic skincare really has rewound the clock on my rapidly ageing face, although a top-up treatment is needed every three to six months to maintain the radiant, youthful and pleasingly natural effect.

Meso-Wharton P199 is available at The Harley Street Skin Clinic from 450 per session (a course of three to six is recommended). Visit harleystreetskinclinic.com or call 020 7436 4441.

Originally posted here:
Tried and Tested: Can Bio Regenerative Skincare really reverse ageing? LLM tried it out - Luxury Lifestyle Magazine

New evidence suggests the use of hormone replacement therapy (HRT) may lead to benefits in certain women with pulmonary hypertension. The findings add to a long debate over the role of hormones like estrogen in the course of the disease.

During a presentation at the American Thoracic Societys 2024 International Conference in San Diego, investigators said HRT appeared to improve pulmonary vascular disease and right ventricular (RV) function in a cohort of 742 women who participated in the study

Corresponding author Audriana Hurbon, M.D., of the University of Arizona College of Medicine, explained along with colleagues that previous research has indicated women with World Symposium Group 1 pulmonary hypertension have improved preservation of RV function compared to men in the same disease group. Yet, Hurbon and colleagues said it was not clear whether the preservation of RV function was linked with endogenous and/or exogenous exposure to female hormones, and it was not known if the apparent benefits of female hormones applied to all groups of pulmonary hypertension or merely to Group 1.

While it is accepted that in World Symposium Group 1 pulmonary hypertension female sex is associated with preservation of right ventricular function, the role of estrogen in pulmonary hypertension has been controversial, Hurbon explained, in a press release. Additionally, we know that women are affected by pulmonary hypertension more often than men, but when compared to each other, women seem to present less severely than men.

The more than 700 participants in Hurbons research were part of the National Heart Lung and Blood Institute-funded Pulmonary Vascular Disease Phenomics (PVDOMICS) Study. The women represented all five World Symposium disease groups, along with healthy controls and comparators who had risk factors for pulmonary hypertension but had not been diagnosed with the disease.

The authors set out to compare participants using mean pulmonary artery pressure on right heart catheterization to measure pulmonary vascular disease related to pulmonary hypertension, and characterizing RV function based on RV fractional shortening and RV ejection fraction from echocardiography.

Endogenous hormone exposure was quantified based on self-reported lifetime duration of menses. Participants were considered to have exogenous exposure to hormones if they had ever received HRT.

Hurbon and colleagues found that people with greater lifetime duration of menses had decreased average pulmonary arterial pressure regardless of which pulmonary hypertension group they belonged in. Specifically, they found mean pulmonary arterial pressure was 4714 mmHg for participants with 20-30 years of menses, versus 3713 mmHg for participants with more than 50 years of menses.

Additionally, participants who had taken HRT had lower mean pulmonary artery pressure (3511 vs 4214, P = 0.002) and pulmonary vascular resistance (53 vs 74, P = 0.006) and higher RV fractional shortening (3711 vs 329, P = 0.001) and RV ejection fraction (4813 vs 4012 %, P < 0.0001). However, when broken out by subgroup, the investigators only found statistically significant impacts in patients with Group 1 pulmonary hypertension.

Hurbon said in the press release that further analysis also suggests that older age and HRT exposure may have a positive synergistic effect.

This could support a theory suggesting a threshold of estrogen exposure necessary for a protective effect, she said.

The authors described their findings as preliminary, but they said their data suggest more research is needed to better understand the potential impacts of HRT, both positive and potentially negative, on patients with pulmonary hypertension.

We hope this study will be a catalyst for further exploration of the mechanisms of female reproductive hormones to identify therapeutic targets for right ventricular preservation in pulmonary hypertension, Hurbon said.

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Hormone Replacement Therapy May Benefit Some Women with Pulmonary Hypertension - Managed Healthcare Executive

Pharmac limits funding to two patches per week but says it is reconsidering this restriction. Photo: BMJ

The shortage of hormone replacement therapy patches is getting worse, and Pharmac has been too slow to act, doctors specialising in women's health say.

Two of the most popular dosage patches - used to mitigate the symptoms of estrogen deficiency - are unavailable nationwide, leaving women shopping around different pharmacies, and some having to pay for extra patches to reach their required prescribed dose.

Pharmac limits funding to two patches per week but said it was reconsidering this restriction, as well as moving to fund alternatives such as estrogen gels.

The funding agency said demand for HRT patches had more than doubled in the past three and the shortage was likely to continue into next year.

There were a number of factors behind the shortage, ERH Associates endocrinologist Dr Megan Ogilvie told Nine to Noon.

"Novartis, which manufactures the gold standard product Estradot, is having manufacturing issues, and there has been a significant rise in demand globally for this product as the superiority of the transdermal approach has been recognised.

"But it is compounded in New Zealand by a lack of gels which would relieve significant pressure on the patches," Ogilvie said.

In a statement, Pharmac said it was "exploring if there are other presentations or products that it can secure and fund".

Ogilvie said more funding for alternatives such as gels would ease the situation.

"We've been asking for funding for gels for probably two to three years now. And it is disappointing to see their comment that they're in the initial stages of procuring funding."

Rules governing the dose pharmacies could dispense were also complicating the situation, Ogilvie said.

"Why if I prescribe 15 micrograms twice weekly and 25 micrograms are the only patches available, does that woman have to pay to make up the extra patch dose?

"It's a historic reason, they [Pharmac] have always only funded two patches per week, per woman."

This means women who needed higher doses were penalised, she said.

Tauranga-based menopause doctor Linda Dear said prescribing and dispensing should be easier and would ease the situation.

"Strictly speaking, the rule is that a woman can have two patches a week of the same strength.

"So, there are pharmacies who will give women say a 25 [microgram] and a 50 [microgram] to make a dose of 75 [micrograms], and not charge for those extra ones."

But the picture was inconsistent, she said.

"Some women have been charged for extra patches, some women are not. Some pharmacists are happy to give women a 100-microgram patch and say 'chop this in half' even if the script said 50 micrograms, some pharmacists are not and they demand a new whole new script from the doctor.

"So, there's all this faffing around, and [at] the centre of this is a poor woman who just needs her HRT and has to go back and forth between doctors and pharmacies, just to just to get the dose she needs."

If it were pills, this would not be an issue, pharmacists could achieve the required dose using various combinations, Dr Dear said.

"It's weird how patch doses are seen so differently, when we just want to achieve a dose in whatever way shape or form, whether you have to give her multiple lower dose, or a higher dose to chop up. Just give her the dose that's working for her."

A consistent message was needed across the board, she said, so that doctors, pharmacists and patients knew what was allowed and what was not.

In a statement, Pharmac chief medical officer Dr David Hughes said he acknowledged the stress of the HRT shortage and that Pharmac was aware some people were paying for extra patches to reach their required dose.

Pharmac was considering reviewing the restriction to two patches per week, he said.

See more here:
Doctors say Pharmac too slow to act over hormone replacement therapy patch shortage - RNZ

Study findings to be presented at the 2024 ASCO Annual Meeting find hereditary risk for gastric and lung cancers, among others, underscoring the need for broader genetic testing

SAN FRANCISCO, May 23, 2024 /PRNewswire/ -- Invitae (OTC:NVTAQ), a leading medical genetics company, today announced eight studies to be presented at the 2024 American Society of Clinical Oncology (ASCO) Annual Meeting held in Chicago from May 31-June 4, 2024. The clinical data being presented demonstrate the importance of genetic testing for patients with various different types of cancers, including breast, gastric, prostate and lung, to better inform management and treatment decisions.

Genetic testing guidelines need to be inclusive of more cancer types, with new data finding gastric, lung and prostate cancer patients with inherited genes linked to increased cancer risk

Gastric cancer is the fourth leading cause of cancer-related deaths worldwide, and the role of pathogenic (disease causing) variants in cancer predisposition genes is not well understood for this disease. One study looked at genetic testing results in 3,706 gastric cancer patients the largest study of its kind to better understand the prevalence of disease causing variants in cancer associated genes. The results found the percentage of patients with disease causing variants to be 13.4%, about 1 in 8 patients. This shows the value of genetic testing in all gastric cancer patients, as the prevalence of pathogenic variants is similar to other cancer types for which guidelines recommend universal genetic testing.

"Current guidelines haven't met the needs for patients across cancer types, gastric cancer included," said Dr. Ophir Gilad, University of Chicago and a co-author of this study. "The prevalence of actionable gene variants found in this study of gastric cancer patients is on par with other cancer types for which guidelines recommend universal genetic testing. We're increasingly seeing evidence for germline genetic testing to help guide treatment plans and familial testing for various cancer types."

Additionally, in a study of 14,317 patients with lung cancer, 12.6% had pathogenic germline variants -- regardless of smoking history. The study results suggest these inherited genes are not only independently associated with lung cancer, but also additive to smoking risk for lung cancer. These data reinforce prior studies supporting consideration of germline genetic testing for all patients with lung cancer, independent of age or reported smoking history.

Genetic testing is similarly underutilized for prostate cancer. In a large study of 15,000 prostate cancer patients that received genetic testing, results showed that of the patients with genetic variants that increase risk of prostate cancer, 3 in 4 patients had no reported family history of prostate cancer and more than 1 in 3 patients had no reported family history of any cancer. The findings underscore the importance of genetic testing for all prostate cancer patients, regardless of age, stage or family history.

Breast cancer data in Rwanda demonstrates need for more genetic testing in underrepresented populations

Despite the observation that cancers are often diagnosed at young ages and take an aggressive course in Sub-Saharan Africa (SSA), genetic data that could inform treatment are limited for this population group.

In a recent study, patients undergoing cancer treatment in hospitals in Rwanda for female breast, male breast and prostate cancer underwent multigene panel testing (Invitae), and the results found a large proportion of the patients had inherited pathogenic variants that could help inform their treatment (18.3% of female breast cancer, 16.7% of male breast cancer, and 4.3% of prostate cancer patients). The findings suggest that genetic testing should be more routinely implemented into cancer care and prevention strategies in this population.

Underrepresented race, ethnicity, and ancestry (REA) groups face these challenges across geographies. In another recent study being presented at ASCO that included more than one million people over an eight-year period who underwent genetic testing for hereditary cancer syndromes, it was found that underrepresented REA groups are disproportionately impacted by variants of uncertain significance (VUS) in genetic testing, which are uncertain results that are not clinically actionable. With more representation of these groups in clinical studies, there will be more data that could uncover life-saving discoveries. Clinical evidence was the most significant source of information leading to VUS resolution, underscoring the importance of the clinician-lab partnership and communication.

"Germline genetic testing should be the standard of cancer care across many types of cancers. In underrepresented populations, this is especially crucial as more information needs to be collected to better inform care and improve population health overall," said Dr. Michael Korn, chief medical officer at Invitae. "Each year, ASCO presents us with an opportunity to share compelling research to help propel cancer treatment forward, and we're proud of the clinical insights our tests are able to provide across cancer types."

Study offers reassurance that variants of uncertain significance in genetic testing results among patients with breast cancer do not lead to overuse of treatment or surveillance interventions, such as mastectomies

It's common for patients with breast cancer undergoing germline genetic testing to have uncertain results, but it's previously been unclear if these results impact clinical management. However, a recent study being presented at ASCO presents new evidence indicating that variants of uncertain significance (VUS) identified through germline genetic testing do not result in guideline-discordant management in real-world settings. Specifically, patients with breast cancer and VUS results demonstrated similar rates of treatment, prevention and surveillance interventions compared to those with negative results. This offers reassurance that VUS results do not lead to overuse of mastectomies or other interventions for patients with breast cancer.

2024 ASCO presentations and posters:

About InvitaeInvitae (OTC: NVTAQ) is a leading medical genetics company trusted by millions of patients and their providers to deliver timely genetic information using digital technology. We aim to provide accurate and actionable answers to strengthen medical decision-making for individuals and their families. Invitae's genetics experts apply a rigorous approach to data and research, serving as the foundation of their mission to bring comprehensive genetic information into mainstream medicine to improve healthcare for billions of people.

To learn more, visit invitae.comand follow for updates on LinkedIn, X, Instagram, and Facebook@Invitae.

Safe Harbor StatementThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, including statements relating to the company's beliefs regarding its new research; the company's belief that its new research demonstrates the importance of genetic testing for many cancers not currently covered by clinical guidelines; the company's belief that its new research demonstrates the need for more research in underrepresented populations; and the company's belief that its research helps move cancer treatment forward.. Forward-looking statements are subject to risks and uncertainties that could cause actual results to differ materially, and reported results should not be considered as an indication of future performance. These risks and uncertainties include, but are not limited to: the applicability of clinical results to actual outcomes; the company's ability to use rapidly changing genetic data to interpret test results accurately and consistently; risks and uncertainties regarding the company's ability to successfully consummate and complete a plan under chapter 11 or any strategic or financial alternative as well as the company's ability to implement and realize any anticipated benefits associated with its sale of assets to Labcorp and the any alternative that may be pursued, including the asset sales and wind down of operations; the company's public securities' potential liquidity and trading; and any impact resulting from the delisting of the company's common stock from the New York Stock Exchange and trading instead on the OTC Pink Marketplace; and the other risks set forth in the company's filings with the Securities and Exchange Commission, including the risks set forth in the company's Quarterly Report on Form 10-Q for the quarter ended September 30, 2023. These forward-looking statements speak only as of the date hereof, and Invitae Corporation disclaims any obligation to update these forward-looking statements.

Invitae PR contact: Renee Kelley [emailprotected]

SOURCE Invitae Corporation

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New Research Demonstrates the Importance of Genetic Testing for Many Cancers Not Currently Covered by Clinical ... - PR Newswire