Archive for the ‘Gene Therapy Research’ Category

INTRODUCTION

Tuberous sclerosis complex (TSC) is a hereditary disease affecting multiple organs with an incidence of about 1 of 5500 (1, 2), resulting from mutations in either TSC1 encoding hamartin or TSC2 encoding tuberin. Hamartin and tuberin normally act as a complex to inhibit mTORC1 (mammalian/mechanistic target of rapamycin complex 1) through guanosine triphosphatase (GTPase) activating effects on Ras homolog enriched in brain (Rheb) (3). When a mutation in the corresponding normal TSC1 or TSC2 allele occurs somatically in susceptible cells, they enlarge and proliferate causing abnormal development and tissue lesions. These secondary mutations can occur prenatally or after birth in different cell types, and the timing and frequency of these hits affect the severity of the disease in a stochastic manner. Neurodevelopmental manifestations are responsible for the greatest morbidity, including severe, refractory epilepsy and hydrocephalus, as well as autism (40%), cognitive impairment (50%), and mental health issues (70%) (46). In addition, renal angiomyolipomas forming later in life can cause life-threatening hemorrhage and/or renal failure, and pulmonary lymphangioleiomyomatosis can severely compromise respiratory function. Current treatments include surgical resection and/or treatment with rapamycin analogs (rapalogs). Although often well tolerated, rapalogs cause immune suppression (7) and potentially compromise early brain development (8), and lifelong therapy is often required. Therefore, there is a clear need to identify other therapeutic approaches for TSC.

Adeno-associated virus (AAV) vectors have been used widely in clinical trials for many hereditary diseases with little-to-no toxicity, long-term action in nondividing cells, and improvement in symptoms (911). Benefit can be seen after a single injection and some serotypes, e.g., AAV9, AAVrh8, and AAVrh10, can efficiently enter the brain, as well as peripheral organs after intravenous (IV) injection (12, 13). The insert capacity of AAV vectors is about 4.7 kb (including promoter, transgene, polyadenylation (poly A) sequence, and other regulatory elements), and the complementary DNA (cDNA) for tuberin (5.4 kb) cannot be accommodated. We generated a cDNA encoding a shorter form of tuberin, termed cTuberin. We tested its lack of toxicity and ability to bind to hamartin and Rheb1, as well as to suppress phosphoS6 kinase activity in cultured cells. In a stochastic mouse model of TSC2 [based on a TSC1 model; (14)], AAV vector encoding Cre recombinase was introduced by intracerebroventricular (ICV) injection into homozygous Tsc2-floxed mice (15) at postnatal day 0 (P0) typically leading to death at about P58 with enlarged ventricles. Near-normal life span and reduction of brain pathology were achieved in most of these animals by a single IV injection of an AAV9 vector encoding cTuberin under a strong, constitutive promoter. These studies demonstrate the ability of cTuberin to suppress overgrowth of tuberin-null cells, including neural cells and, presumably, other cells in the body, and, hence, support the preclinical efficacy of AAV-cTuberin for TSC2 lesions.

Whereas hamartin is encoded in a cDNA of 3.5 kb, which fits into an AAV vector (16), the cDNA for tuberin (5.4 kb) is too large. To generate a potentially functional form of tuberin encoded in a shorter cDNA, we retained the N-terminal domain that binds to hamartin and the C-terminal domain containing GAP (GTPase-activating protein) activity that inhibits Rheb, with N-terminal region and phosphorylation of the C-terminal region of tuberin also thought to regulate formation of the complex with hamartin Fig. 1A (3, 1720). The potential for cTuberin to retain some functional activity was supported by findings of Momose et al. (21) that genomic overexpression of the C-terminal region of rat tuberin (amino acids 1425 to 1755) can suppress renal tumors in the Tsc2 Eker rat model. We felt it was also important to retain the hamartin-binding domain at the N terminus, as hamartin and tuberin function together as a complex with Tre2-Bub2-Cdc16 (TBC) 1 domain family, member 7 (TBC1D7) to accelerate guanosine triphosphate (GTP) to guanosine diphosphate conversion of Rheb-GTP (3, 22). In addition, this requirement for complex formation for activity might act to limit potential negative effects of high levels of transgenic cTuberin expression. cTuberin was thus designed to retain key elements of function, including 450 amino acids from the N-terminal region and 292 amino acids from the C-terminal region, joined by a flexible serine-glycine linker of 16 amino acids (fig. S1). This cDNA, with a Kozak sequence, and a C-terminal c-Myc tag was inserted into an AAV2 backbone under a chicken -actin (CBA) promoter (23), with a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) and poly A signals (Fig. 1B).

(A) The functional domains of tuberin are depicted with numbers representing amino acid residues for the full-length human proteins [based on (3)]. T1BD, hamartin-binding domain; GAP, GAP domain homologous with that in Rap1GAP. cTuberin contains the T1BD and GAP domains of TSC2 with a glycine-serine linker and C-terminal c-Myc tag. (B) Schematic of AAV-cTuberin transgene expression cassette. ITR, inverted terminal repeats; CBA, chicken -actin promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; pA, poly A signal sequences [from SV40 and bovine growth hormone (BGH)].

Human embryonic kidney (HEK) 293T cells were transfected with plasmids for empty AAV (AAV1-null that contains all the elements except the cTuberin cDNA), AAV-CBAgreen fluorescent protein (GFP), or AAV-CBA-cTuberin-Myc to assess the expression level of cTuberin. In addition to endogenously expressed tuberin (200 kDa), cTuberin expression at the appropriate molecular weight (MW) of 85 kDa was detected on Western blots using anti-tuberin and anti-Myc antibodies (Fig. 2A; representative blot, n = 3) Immunocytochemistry of 293T cells transfected with different plasmids demonstrated stronger tuberin immunoreactivity in those transfected with AAV-CBA-cTuberin-Myc compared to other groups that expressed only endogenous tuberin (Fig. 2B; representative micrographs, n = 3). We determined transfection efficiency of these cells in two waysmicroscopically and by flow cytometry. As it is challenging to differentiate expression of endogenous tuberin from cTuberin, image analysis was carried out microscopically for each well (approximately 2000 cells per well; n = 3) for 4,6-diamidino-2-phenylindole (DAPI)positive and c-Mycpositive cells, and we determined that 43 2% of cells were transfected with the AAV-CBA-cTuberin-Myc (Fig. 2B). Cytotoxicity assays were also performed following transfection of HEK293T cells with AAV-null, AAV-CBA-GFP, or AAV-CBA-cTuberin-Myc plasmids to evaluate potential toxicity of cTuberin. The lactate dehydrogenase (LDH) assay (Dojindo Molecular Technologies Inc., Rockville, MD, USA) revealed no cytotoxicity in cTuberin-transfected cells, as compared to controls (Fig. 2C; n = 3). As a second way to evaluate the extent of transfection of these 293T cells with AAV-CBA-cTuberin-Myc plasmid DNA (n = 3), we sorted the c-Mycpositive cells using flow cytometry, after staining the cells with unlabeled c-Myc primary antibody followed by Alexa Fluor 647conjugated secondary antibody. Compared to the background in nontransfected cells (4 1%), we detected a marked increase of c-Mycpositive cells (50 1% or 46% minus the background, similar to the 43% determined by cell counting) after transfection with the AAV-CBA-cTuberin-Myc plasmid (P < 0.0001) (Fig. 2D). This suggests that the apparently endogenous levels of cTuberin reflect 43 to 46% transfection efficiency and that levels of cTuberin are about twice as high as endogenous tuberin in these transfected cells, without apparent toxicity.

(A) HEK293T cells were transfected with empty AAV (AAV-null), AAV-CBA-GFP, or AAV-cTuberin-Myc (AAV-CBA-cTub-Myc) plasmids. Representative Western blot (WB) (from n = 3 experiments) shows endogenous tuberin (~200 kDa) using anti-tuberin antibody and cTuberin-Myc (predicted 85 kDa) using anti-tuberin and anti-Myc antibodies. -Actin served as a loading control. (B) HEK293T cells were transfected with AAV-null and AAV-cTub-Myc plasmids and immunostained 72 hours later for tuberin (red) and c-Myc (green) with nuclear DAPI (blue). Scale bar, 100 m. The bar graph (bottom right) summarizes the cell count analysis (43 2% of the AAV-cTuberin-Myctransfected cells expressed c-Myc). (C) Cell death was quantified 72 hours after transfection using the Cytotoxicity LDH Assay Kit. Each bar represents the mean SD. (n = 3). ****P < 0.0001, compared with the positive apoptotic control (Bortezomib, 100 nM). (D) To further quantify transfection efficiency, HEK293T cells were transfected with AAV-CBA-cTub-Myc plasmid for 72 hours (n = 3 experiments) followed by sorting for the c-Mycpositive cells using flow cytometry. There was a significant increase in c-Mycpositive cells (50 1%) in the transfected cells (P < 0.0001) as compared to the nontransfected cells (4 1%). ****P < 0.0001.

COS-7 cells were cotransfected with plasmids for empty AAV (AAV-null), Myc-tagged full-length tuberin (Myc-FL-tuberin), AAV-CBA-cTuberin-Myc, Myc-tagged glycogen synthase kinase-3 (Myc-GSK-3), FLAG-tagged hamartin, and/or hemagglutinin (HA)tagged glutathione S-transferase (GST)tagged Rheb1 (HA-GST-Rheb1). Coimmunoprecipitation experiments performed with anti-Myc antibody showed that Myc-tagged cTuberin bound to FLAG-tagged hamartin and HA-tagged GST-Rheb1 to the same extent as Myc-FL-tuberin (Fig. 3). Myc-tagged GSK-3, used as a negative control, did not bind to FLAG-tagged hamartin or HA-tagged GST-Rheb1. These results indicated that cTuberin binds to hamartin and Rheb1 in cells, supporting a similarity in these biochemical parameters between cTuberin and full-length tuberin.

Representative blot (n = 3 experiments) after cotransfection of the Myc-tagged cTuberin (AAV-CBA-cTub-Myc) or full-length tuberin (Myc-FL-tuberin) along with FLAG-tagged hamartin and HA-tagged GST-Rheb1. Coimmunoprecipitation (co-IP) using anti-Myc antibody demonstrated that cTuberin-Myc interacts with both Flag-hamartin and HA-Rheb1 similar to Myc-FL-tuberin. Conversely, negative control Myc-GSK-3 showed no interaction with FLAG-hamartin or HA-GST-Rheb1.

For functional assessment tuberin and cTuberin on mTORC1 activity in vitro, we evaluated S6K T389 phosphorylation in cells expressing these proteins, together with hamartin and S6K, as described (24, 25). To determine whether cTuberin overexpression could inhibit mTORC1 activation, the Myc-tagged cTuberin plasmid was cotransfected with Flag-tagged hamartin and HA-tagged p70S6K (HA-S6K) reporter plasmids into HEK293T cells. As a control, a plasmid encoding Flag-tagged full-length tuberin was cotransfected with Flag-hamartin and HA-S6K plasmids. Hamartin and full-length tuberin coexpression inhibited phosphorylation of S6K T389, as expected, and similarly, coexpression of hamartin and cTuberin also decreased pS6K T389 levels (Fig. 4A), supporting the ability of cTuberin to bind to hamartin and efficaciously inhibit TORC1 activity. Level of pS6K T389 inhibition was quantified relative to HA-S6K using Fiji/ImageJ. Flag-hamartin cotransfected with AAV-GFP served as a control (normalized to 1.0), and cotransfection with full-length tuberin and cTub-Myc revealed a significant inhibition of S6K T389 phosphorylation by 69 and 56%, respectively (*P < 0.05; n = 3 separate experiments).

(A) Full-length Flag-tagged tuberin (Flag-tuberin), Myc-tagged cTuberin (AAV-cTub-Myc), or AAV-GFP plasmids were cotransfected into HEK293T cells along with full-length Flag-tagged hamartin (Flag-hamartin) and HA-tagged p70S6K (HA-p70S6K), which is phosphorylated at T389 by mTORC1 (latter used as a reporter for mTORC1 activation). Representative blot (n = 3 experiments) demonstrated similar inhibition levels of phosphorylated p70S6K (pS6K T389) with either full-length tuberin or cTub-Myc cotransfected with full-length hamartin. (B) Quantitation of decrease in S6K T389 phosphorylation was performed relative to HA-S6K using Fiji/ImageJ. Flag-hamartin cotransfected with AAV-GFP served as a control (normalized to 1.0), and cotransfection with full-length tuberin or cTub-Myc revealed inhibition of 69 or 56%, respectively, representing the results from three experiments. *P < 0.05.

To evaluate preclinical efficacy of the AAV9-CBA-cTuberin-Myc vector (hereafter referred to as AAV9-cTuberin), Tsc2 homozygous floxed mice (referred to as Tsc2-floxed or Tsc2flox) were first injected ICV at P0 with an AAV1-CBA-Cre recombinase vector (1 1012 vg/kg) to inactivate Tsc2 in a subset of neurons, astrocytes, and other cells in the brain (16). At P21, these AAV1-Creinjected mice were injected IV (retro-orbitally) with AAV9-cTuberin vector (9 1011 vg/kg) or AAV9-null vector (1 1013 vg/kg) and were compared to control animals that did not receive any IV injection. Tsc2-floxed AAV1-Creinjected (P0) mice had a median survival of 58 days, as did similar mice injected IV at P21 with AA9-null vector (mean survival of 58 days), mice injected IV at P21 with AAV9-cTuberin vector had the median survival of 462 days (P < 0.0001) (Fig. 5A). We also tested the potential toxicity of this dose of AAV9-cTuberin alone by injecting six Tsc2-floxed mice IV at P21 (in the absence of AAV1-Cre induced loss of Tsc2 at P0). All six mice survived over 500 days without apparent toxicity (Fig. 5A).

(A) Tsc2-floxed mouse pups were injected ICV with an AAV1-Cre vector (1 1012 vg/kg) at P0 to induce tuberin loss in multiple cell types in the brain. At 21 days, mice were injected IV with either AAV9-cTuberin (9 1011 vg/kg; n = 12) or AAV9-null (1 1013 vg/kg; n = 6) or noninjected (n = 6). Median survival of the AAV-cTuberin-injected mice (462 days, red line) was significantly longer than the non-cTuberin-injected mice (58 days, black line) (****P < 0.0001). Mice injected secondarily with the AAV9-null vector also died on average by 58 days (gray). Pups injected only with AAV9-cTuberin (no AAV1-Cre) all lived over 500 days. For (B) and (C), AAV1-Cre ICV (1 1010 vg/kg) was injected at P1 only or followed with AAV9-cTuberin (8 1012 vg/kg) IV at P21. (B) Body weights of Tsc2-floxed mice injected with AAV1-Cre vector, with and without AAV9-cTuberin vector, or noninjected were similar from P21 to P50. (C) For the rotarod test, the motor function of the Tsc2-floxed AAV1-Creinjected mice rescued by AAV9-CBA-cTuberin vectors was significantly better than that of the AAV1-Cre group and noninjected group. **P < 0.005. ns, not significant.

Different cohorts of mice were subjected to body weight measurement and motor function assessment starting at P21/22 for nave, noninjected animals, AAV1-Cre ICV injected (1 1010 vg/kg) at P1 only or followed with AAV9-cTuberin injected (8 1012 vg/kg) IV at P21. Body weights of these mice from age 21 to 50 days did not differ according to treatment (Fig. 5B). Movement was assessed using an automated rotarod apparatus with accelerating rotary velocity (4 going to 64 rpm over 2 min) to assess motor skills of the mice as time of latency to fall. A significant increase in latency was observed for the AAV1-Cre + AAV9-cTuberin as compared to the AAV1-Creinjected mice and naive mice (Fig. 5C). During animal handling, two mice of six Tsc2-floxed AAV-Creinjected mice (day 41) and two mice of seven Tsc2-floxed AAV-Creinjected + AAV-cTuberininjected mice (one each on days 47 and 50) manifested straub (vertical tail), humped back, and/or motor seizures, which did not, however, compromise their consequent rotarod performances (fig. S2).

Two other approaches were less effective at extending survival of AAV1-Cre ICVinjected Tsc2-floxed mice. In one, using a similar time scheme (fig. S3), Tsc2-floxed pups were injected with 1 1014 vg/kg AAV1-Cre ICV at P3 and then 3 1012 vg/kg of AAV1-cTuberin (in contrast to AAV9 serotype) IV at P21, with the higher amount of AAV1-Cre (without cTuberin) leading to death with a mean of 36 days and survival only being extended by AAV1-cTuberin to a mean of 54 days. This probably reflects the fact that AAV1 is less efficient at crossing the blood-brain barrier (BBB) than AAV9. In another experiment, the Tsc2-floxed pups were injected ICV with AAV1-Cre (1 1012 vg/kg) at P0, followed by ICV injection (in contrast to systemic injection) of 4.5 1013 vg/kg of AAV9-cTuberin at P3. This approach led to median survival of 50 days in Tsc2-floxed mice without cTuberin injection, while those injected with AAV9-cTuberin had extended median survival only up to 95 days (fig. S4). This experiment raises the possibility that other lesions in the body (in addition to the brain) resulting from ICV injection of AAV1-Cre were associated with death and were not sufficiently alleviated by ICV injection of the cTuberin vector and/or that the high dose AAV-cTuberin injected ICV into P3 pups had some toxicity (26).

In nave (normal) Tsc2-floxed mice, the ventricle is lined by a single layer of ependymal cells (Fig. 6A). Neuropathological examination at P42 revealed that ICV injection of AAV1-Cre in Tsc2-floxed mice at P0 led to multiple layers of ependymal and subependymal cells lining the lateral ventricle (indicating increased proliferation of these cells) (Fig. 6B, asterisk), which sometimes appeared as nodules along the ventricular lining (Fig. 6C). When these AAV1-Creinjected mice were treated with AAV9-cTuberin (IV injected at P21), there was apparent regression of ependymal/subependymal overgrowths (Fig. 6D). We also stained these mouse brain sections (P42) for Ki67 as an indication of cell proliferation. As expected, there was little-to-no proliferation of ependymal/subependymal cells lining the ventricles in the nave brain (Fig. 7A). In contrast, after AAV1-Cre injection at P0, there was marked proliferation of these cells, including apparent migration of dividing cells into the brain parenchyma (Fig. 7B), also seen after subsequent IV injection with AAV9-null vector (Fig. 7C). In contrast, IV injection of the AAV9-cTuberin vector decreased proliferation and inward migration of Ki67+ ependymal/subependymal cells (Fig. 7D).

Tsc2-floxed mouse pups were either not injected (nave) or injected ICV in both ventricles (1 1012 vg/kg) with an AAV1-Cre vector at P0. At 21 days, some mice were injected IV with AAV9-cTuberin (9 1011 vg/kg) or noninjected. At 42 days, all mice were euthanized. (A) Nave, noninjected brain (black arrowhead indicating the choroid plexus). (B and C) Tsc2-floxed mice with AAV1-Cre at P0 and no further injection showed (B) proliferation of ependymal/subependymal cells (asterisk) and (C) subependymal nodules. (D) Little-to-no subependymal overgrowth was detected in mice receiving both the P0 AAV1-Cre ICV injection and P21 IV AAV9-cTuberin injection. Representative images are shown. Magnification bar, 100 m. CC, corpus callosum; LV, lateral ventricle.

Tsc2-floxed mouse pups were either not injected (nave) or injected ICV in both ventricles (1 1012 vg/kg) with an AAV1-Cre vector at P0. At 21 days, some mice were injected IV with AAV9-cTuberin (9 1011 vg/kg), AAV9-null (1 1013 vg/kg) or noninjected. At 42 days, all mice were euthanized. (A) Nave, noninjected brain reveals little-to-no staining in the ependymal/subependymal layers. (B) Tsc2-floxed mice injected with AAV1-Cre vector only showed abnormal mitotic activity and apparent migration of cells (yellow arrows) away from the ventricular zone, as well as multiple ependymal/subependymal layers (green arrowheads) as compared to the nave group. (C) Tsc2-floxed mice injected with AAV1-Cre vector followed by AAV9-null vector showed abnormal mitotic activity of the cells and thickening of the subventricular zone. (D) The Tsc2-floxed mice injected with AAV1-Cre and then rescued with the AAV9-cTuberin vector showed a trend toward normalization of the ependymal/subependymal layer. The corresponding brain sections were counterstained with DAPI. The yellow asterisk denotes autofluorescence in the choroid plexus. Representative images are shown. Magnification bar, 100 m.

The brain sections (P42) were also immunostained for phosphorylated ribosomal protein S6 (pS6). We observed low pS6 expression in the whole brain sections of the noninjected (nave) mouse brain (Fig. 8A, top). In contrast, in AAV1-Cre ICVinjected Tsc2-floxed mice, pS6 expression was intense in many brain cells [Fig. 8, A (middle) and Bi], with the pS6-positive cells being significantly larger in size (Fig. 8Bii) and with a higher pS6 immunofluorescence signal (Fig. 8Biii). When the AAV1-Creinjected mice were subjected to IV injection of the AAV9-cTuberin vector at P21, the pS6 immunoreactive cells were significantly decreased in average size by 23% [P < 0.05; Fig. 8, A (bottom) and Bii] and showed a reduced pS6 signal by 28% (P < 0.05; Fig. 8Biii) consistent with reduced mTOR activity.

Tsc2-floxed mouse pups were either not injected (nave) or injected ICV (1 1012 vg/kg) with an AAV1-Cre vector at P0. At P21, some mice were injected IV with AAV9-cTuberin (9 1011 vg/kg) or noninjected. All were euthanized at P42. (A) Whole mouse brain sections from nave, AAV1-Cre, and AAV1-Cre+ AAV9-cTuberin injected mice stained for pS6 and DAPI. Representative whole brain sections (scale bar, 1 mm; eight-bitthresholded inverted images) indicated absence of pS6 puncta in nave group. In other groups, pS6 puncta appeared as darkened spots within the cerebral cortex and caudate putamen; high magnification inset images (scale bar, 100 m; 12-bitthresholded inverted images). (B) pS6 analysis included puncta density (i), size (ii), and intensity (iii). *P < 0.05; n = 3. a.u., arbitrary units. (C) Compared to nave pups, immunoblotting demonstrated AAV1-Cremediated decrease of tuberin (54%) and increase in pS6 (76%) in Tsc2-floxed mice injected with AAV1-Cre, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with nave brain as control (normalized to 1.0; *P < 0.05; n = 3). (Di) Ct values for biodistribution of AAV vector genomes in the brain and liver measured by qPCR. (Dii) Ct value of GAPDH and cTuberin cDNAs in brains of nave animals injected with AAV1-Cre only or with AAV1-Cre and AAV9-cTuberin. n.d., not determined.

To assess the Cre-mediated loss of tuberin and activation of mTOR activity in vivo, newborn pups (P0, n = 3) were injected ICV with AAV1-CBA-Cre recombinase vector (AAV1-Cre at dosage of 1 1012 vg/kg), and another three noninjected (nave) pups were included as controls. One week after injection of vector, brain protein lysates were collected for immunoblotting with anti-tuberin and anti-pS6 antibodies. There was significant reduction in expression of tuberin by 54% (P < 0.05) and significant increase of pS6 by 76% (P < 0.05) in animals injected with AAV1-Cre, confirming that the Cre recombinase mediates loss of tuberin and activation of mTOR in the treated mice (Fig. 8C).

To examine the vector biodistribution in the injected animals, Tsc2-floxed animals were injected ICV at P0, with an AAV1-CBA-Cre recombinase vector (AAV1-Cre; 1 1012 vg/kg, n = 4). At P21, these AAV1-Creinjected mice were injected IV with AAV9-cTuberin vector (9 1011 vg/kg). One week after injection, DNA was extracted from the brain and liver of these animals. For comparison, another three Tsc2-floxed animals subjected to no injections were used as controls. For quantitative polymerase chain reaction (qPCR) analysis of AAV genomes (probes and primer specific), 50 ng of DNA was used as a template, and primers and probes were designed to amplify the cTuberin in the infected animal (fig. S5). cTuberin DNA was not detected in the noninjected control group (Fig. 8D). Cycle threshold (Ct) values for the Tsc2-floxed animals injected with AAV1-Cre and AAV9-cTuberin vectors were readily detectable with approximately 30.8 2.6 and 17.2 0.2 cycles for brain and liver tissue, respectively (Fig. 8Di). The large difference between the AAV genomes in brain compared to liver is likely due to both the high tropism of systemically injected AAV for the liver and the relatively low dose of vector injected (9 1011 vg/kg). To detect cTuberin transgene expression, total RNA was extracted from the brains and livers of another set of animals, including noninjected controls; Tsc2-floxed animals injected with AAV1-Cre only, and Tsc2-floxed animals injected with AAV1-Cre and AAV9-cTuberin vectors (n = 3 for all groups), with the dosage of AAV1-Cre ICV injected at P1 (1 1010 vg/kg) or combined with AAV9-cTuberin injected IV at P21 (1.8 1012 vg/kg). Quantitative reverse transcription PCR (RT-qPCR) analysis indicated that cTuberin mRNA was undetectable in the noninjected control group and those injected with AAV1-Cre only. In contrast, in both brains and livers, we detected cTuberin mRNA in mice injected with AAV9-cTuberin at levels of Ct 36.8 3 and 34.8 0.5 cycles, respectively (Fig. 8Dii). We did not detect cTuberin cDNA when reverse transcriptase was omitted from the RT reaction, indicating that we were detecting bona fide cTuberin mRNA and not sample contamination with AAV-cTuberin genomes.

This is the first description of an alternative mode of therapy for TSC type 2 (TSC2) involving gene replacement using an AAV vector encoding a condensed form of tuberin, termed cTuberin. We developed a stochastic mouse model for central nervous system (CNS) lesions in TSC2 in which homozygous Tsc2-floxed mice (15, 27) are injected ICV in the newborn period (P0 to P3) with an AAV1 vector expressing Cre recombinase, as described for our stochastic TSC1 model (14). AAV1-Cre injection in the Tsc2-floxed model resulted in death at about 58 days. Death appeared to be due primarily to hydrocephalus caused by ependymal/subependymal overgrowths blocking cerebrospinal fluid flow, with whole-body pathology revealing no overt lesions except in the CNS. Although signs of seizures were noted in a few mice during motor performance assessment, these animals recovered normal activity. Experiments showed that IV injection of AAV9-cTuberin vector into this stochastic Tsc2-floxed mouse model on day 21 extended life span in most mice (9 of 12) to at least 450 days. Histochemical/immunohistochemical analysis of the brains supported a resulting reduction in size of ependymal/subependymal lesions, decreased proliferation of cells in the subependymal zone, and reduced phosphorylation of S6 kinase driven by mTOR activity. This study offers a potential single treatment paradigm for improving the outcome of patients with TSC2.

Limitations to this stochastic Tsc2 mouse model include the fact that floxed alleles (before Cre exposure) are normal in function during prenatal development and that Cre recombinase usually knocks out both alleles in a cell at once, which is different from the case in TSC2 patients, most of whom are heterozygous for one mutant and one normal allele in most-to-all cells in their body. TSC2 heterozygosity itself may compromise some cell functions and contribute to aspects of the disease phenotype (1, 28, 29). Further, the model used here is CNS oriented, with most pathology in the brain; whereas in TSC patients, a number of organs in addition to the brain are affected. In addition, this Tsc2 mouse model does not show all the brain abnormalities observed in human TSC2, many of which form prenatally, such as cortical tubers, disorganized cortical lamination, dysplastic neurons, and giant cells (30). Strengths of this model are that there is loss of tuberin expression in a number of different cell types in the brain with variation for animal to animal, as occurs in patients with TSC. This is in contrast to commonly used models where Tsc2-floxed mice are mated to mice expressing Cre recombinase under a cell-specific promoter, e.g., the synapsin promoter, in which case most and only neurons lose expression at embryonic day 12.5 (31).

The central portion of tuberin that was removed to fit coding sequences into the AAV vector contains a number of phosphorylation sites that are involved in regulating mTOR activity under some circumstances, with three of these sites bearing missense mutations associated with TSC2, suggesting that they may contribute to the disease phenotype or create truncated, nonfunctional proteins (6). By comparison, there is an ortholog of human tuberin in Schizosaccharomyces pombe that lacks about 500 amino acids in the equivalent central region of human tuberin, suggesting that these sites are dispensable to some functions (32). Further, some of the key Akt phosphorylation sites in mammalian tuberin are not essential in Drosophila (33), and phosphorylation sites for Akt, ribosomal protein S6 kinase, and AMP-activated protein kinase (AMPK) in the central region of human tuberin are not present in Schizosaccharomyces or Dictyostelium (34), suggesting that these sites may not be critical for function. Given the critical role of phosphorylation sites in tuberin in growth factor and cytokine signaling in mammalian cells, one would anticipate that cTuberin in TSC2-null cells would lack some of these regulatory controls. However, in the Eker rat model of TSC2, which is prone to renal carcinomas, the C-terminal region alone (amino acids 1425 to 1755) of rat tuberin suppresses tumor formation in a dose-dependent manner (35). Fortunately, in TSC2 patients, only a very small fraction of cells in the body suffer loss of tuberin, and most damage is done by the enlargement and proliferation of these deficient cells. Thus, if overgrowths can be suppressed by cTuberin, then that would bring therapeutic benefit for many of the symptoms of the disease, although the cells would not be fully normalized. So far, in cultured cells, cTuberin has been shown to bind to hamartin, and overexpression of cTuberin was not found to be toxic. cTuberin inhibited mTORC1 signaling in these cells to the same extent as tuberin, supporting the use of cTuberin as an effective replacement for tuberin for some cell properties.

Subependymal nodules (SENs) occur in 10 to 15% of children with TSC, usually appearing after birth and being more severe in TSC2 than TSC1 (3638). SENs can enlarge into subependymal giant cell astrocytomas (SEGAs) during the first decade of life causing obstruction of cerebrospinal fluid flow, potentially leading to life-threatening hydrocephalus, as well as endocrinopathy and visual impairment (36, 37, 39, 40). Under optimal care, infants and children with TSC are monitored for subependymal lesions by magnetic resonance imaging (MRI) every 6 to 12 months. The two current standards of care are neurosurgical removal of SEGAs through craniotomy, which can be associated with significant morbidity (37), or treatment with rapalogs, which inhibit mTOR activity. Rapalogs have proven effective in reducing lesion size, but they require continuous treatment and have limited access to the brain after peripheral administration. Potential problems with this class of drugs include a compromise of immune function (41), interference with white matter integrity (42), and possible interference with brain development in early childhood (43). In several studies, the mTOR pathway has been found to be critical to neurodevelopment, including neuronal growth, axonal guidance, synapse formation, and myelination (4446). Inhibition of mTOR by rapalogs may contribute to the observed memory dysfunction following prenatal/postnatal drug treatment in Tsc mouse models (47) and the behavioral abnormalities in wild-type mice treated prenatally with rapamycin (48). Some physicians do not recommend the use of these drugs in children or pregnant women as long-term effects on growth and development in pediatric patients are not fully known (43). Although in at least one study, rapalog treatment was reported to have no significant effect on neurocognitive function or behavior in children with TSC (49).

Our premise is that current therapies for children with TSC may have associated morbidity resulting in the potential for decreased mental functions. Another therapeutic approach would be intravascular administration of an AAV vector that can cross the BBB encoding a replacement gene for the mutant TSC1 or TSC2 alleles. Since SENs are slow growing, there would be time to monitor their size by MRI over several months and leave open the opportunity to administer standard-of-care treatment, as needed. It is hoped that gene replacement therapy might reduce use of more problematic standard-of-care procedures in young children and provide long-lasting benefit with a single administration. Certain serotypes of AAV, such as AAV9, are able to penetrate the BBB as well as deliver to peripheral tissues (13). Thus, with IV delivery, extra copies of the replacement gene would be provided to multiple tissues, including brain, kidney, liver, and lungs, which might reduce the likelihood that somatic mutations in TSC genes later in life would lead to disruptive hamartomas.

Advantages of AAV gene therapy are the potential for a single vector injection yielding long-term transgene expression in nondividing cells. It is assumed that once a tuberin analog is delivered to cells in TSC2 lesions, they would shrink and stop dividing and, hence, retain transgene expression. Gene therapy may be a viable option for infants/children with TSC to reduce potential compromise of brain functions caused by congenital lesions and secondary sequelae of these lesions. AAV9 vectors have been used in young mice with spinal muscular atrophy (SMA) for gene replacement of the survival motor neuron (SMN) protein using both IV (50) and intrathecal (51) gene delivery. An AAV9-SMN drug, Zolgensma (Novartis), is now U.S. Food and Drug Administrationapproved for IV treatment of babies/children with SMA. Two critical aspects of successful gene therapy with AAV vectors are as follows: (i) a known target, in the case of TSC2 loss of function of tuberin; and (ii) no toxicity resulting from overexpression of the replacement protein, since levels of expression cannot at present be regulated. There is a predicted reduced chance of toxicity of cTuberin as it should only be active in a 1:1 complex with hamartin, and hamartin levels are normal in TSC2 null cells (52), with cTuberin not bound to hamartin presumably being degraded. So far, no toxic effects of cTuberin expression have been observed in cells in culture or in mice. Clinical trials should be facilitated by the ability to image reduced lesion size within months by MRI due to shrinking of cell volume and inhibition of cell proliferation, as was found in the rapalog trial for renal angiomyolipomas (53). Typically, AAV vectors are just administered once due to previous exposure to the AAV virus in life eliciting an immune response to the capsid and reducing secondary transduction (54). If replacement is insufficient to reduce symptoms or new TSC2 null lesions arise later in life after AAV gene replacement, it would still be possible to treat patients with rapalogs or possibly exoAAV (55). These studies support the potential of AAV gene therapy for TSC2, which might be especially useful in infants and children where drug inhibition of the mTOR pathway may interfere with early brain development.

The AAV vector plasmid, AAV-CBA-Cre-BGHpA, was derived as described in Prabhakar et al. (16). These AAV vectors carry AAV2 inverted terminal repeat elements, and gene expression is controlled by a hybrid promoter (CBA) composed of the cytomegalovirus (CMV) immediate/early gene enhancer fused to the -actin promoter (23). To increase the efficiency of cTuberin translation (for future use in human gene therapy approach), cDNA encoding cTuberin was human codon-optimized before gene synthesis by GenScript Biotech (Piscataway, NJ, USA). AAV vector plasmid, AAV-CBA-cTuberin-c-Myc, was derived from the plasmid pAAV-CBA-W (56). This vector contains the CBA promoter driving cTuberin, followed by a WPRE and both SV40 and bovine growth hormone (BGH) polyadenylation (poly A) signal sequences. Our cTuberin construct contains the following: ACC (Kozak sequence) :: amino acids 1 to 450 of human tuberin::gly/ser linker :: amino acids 1515 to 1807 of human tuberin :: c-Myc tag = 2307 bp encoding an 85-kD protein (fig. S1). The pAAV-CBA-W, which contains the CBA promoter, WPRE, and poly A sequences, but no transgene, served as AAV-null in our studies.

AAV1 and AAV9 serotype vectors were produced by transient cotransfection of HEK293T cells by calcium phosphate precipitation method of vector plasmids (e.g., AAV-CBA-cTuberin-Myc), adenoviral helper plasmid pAdF6, and a plasmid encoding AAV9 (pAR9) or AAV1 (pXR1) rep and capsid genes, as previously described (57). All AAV vectors carried the identity of all PCR-amplified sequences as confirmed by sequencing. Briefly, AAV vectors were purified by iodixanol density gradient centrifugation. The virus-containing fractions were concentrated using Amicon Ultra 100-kDa molecular weight cut-offs (MWCO) centrifugal devices (EMD Millipore, Billerica, MA, USA), and the titer vector genomes (vg) per milliliter was determined by quantitative real-time PCR amplification with primers and TaqMan probe specific for the BGH poly A signal.

HEK293T cells [American Type Culture Collection (ATCC)] and COS-7 cells (ATCC, Manassas, VA, USA) were cultured in Dulbeccos modified Eagles medium (DMEM; Thermo Fisher Scientific, Hampton, NH, USA) supplemented with 10% fetal bovine serum (FBS; Gemini Bio Products, West Sacramento, CA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific). The cell cultures were periodically screened to ensure they are free from mycoplasma contamination using the PCR Mycoplasma Detection Kit (ABM, G238, Richmond, BC, Canada).

HEK293T cells were seeded in 96-well plates (10,000 cells per well) and, after 24 hours, transfected with various plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 250 ng/10,000 cells using Lipofectamine 2000, according to the manufacturers instructions (Life Technologies, Carlsbad, CA, USA) in Opti-MEM (Life Technologies). Six hours later, transfection media was removed and replaced with DMEM (10% FBS and 1% Penicillin-Streptomycin solution), and cells were allowed to grow for 72 hours. One group of cells was treated with potent proteasome inhibitor Bortezomib (VELCADE; Millennium Pharmaceuticals Inc., Cambridge, MA, USA) (58) at 250 nM for 72 hours, as a positive control for toxicity. Cellular toxicity caused by plasmid DNA transfection was assessed by quantification of extracellular LDH activity using LDH assay kit-WST (Dojindo Molecular Technologies Inc.), following the manufacturers instructions. Briefly, the supernatant for each transfected or treated sample was collected and incubated with substrate for 30 min at 37C. Following incubation, stop solution was added, and absorbance was measured at 490 nm.

Briefly, cultured cells were harvested in lysis buffer [50 mM Hepes (pH 8.0), 150 mM NaCl, 2 mM EDTA, 2.5% sodium dodecyl sulfate, 2% CHAPS, 2.5 mM sucrose, 10% glycerol, 10 mM sodium fluoride, 2 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA), 10 mM sodium pyrophosphate, and protease inhibitor cocktail (P8340, Sigma-Aldrich)]. After sonication and incubation at 8C for 10 min, the samples were centrifuged at 14,000g for 30 min at 8C. Equal amounts of protein, determined by a detergent-compatible protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), were boiled for 5 min in Laemmli sample buffer (Bio-Rad), separated by SDSpolyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membranes (Bio-Rad). Equal protein loading was confirmed by Ponceau S staining. The membranes were blocked in 2% blocking reagent (GE Healthcare, Pittsburgh, PA, USA) for 1 hour at room temperature (RT) and incubated with primary antibodies overnight at 4C. Anti-tuberin/TSC2 (#3612), antiphospho-S6 (#2211), anti-S6 (#2212), anti-Myc (clone 9B11, #2276) (Cell Signaling Technology, Danvers, MA, USA), anti-actin (#A5441), anti-FLAG (clone M2, #F1804) (Sigma-Aldrich), anti-HA (clone F-7, sc-7392, Santa Cruz Biotechnology, Dallas, TX, USA), and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#CB1001, EMD Millipore) were used as primary antibodies. Anti-rabbit or anti-mouse immunoglobulin G antibody conjugated with horseradish peroxidase was used as a secondary antibody (Thermo Fisher Scientific). Enhanced chemiluminescence reagent, Lumigen ECL Ultra (TMA-6) (Lumigen, Southfield, MI, USA), was used to detect the antigen-antibody complexes.

For immunoprecipitations, COS-7 cells were transfected with plasmid vectorsAAV empty, AAV-CBA-cTuberin-Myc, pcDNA-hamartin-FLAG (V. Ramesh laboratory), pReceiver-M09/tuberin-Myc (catalog no. EX-Z5884-M09, GeneCopoeia, Rockville, MD, USA), pCMV-Tag3A-Myc-GSK-3 (GSK-3 sequence was cloned into pCMV-Tag3A vector; catalog no. 211173-51, Agilent Technologies, Santa Clara, CA, USA), and pRK5-HA-GST-Rheb1 [catalog no. 19310, Addgene, Watertown, MA, USA; provided by Sancak et al. (59)] using Lipofectamine 2000 (Life Technologies). Cells were lysed with ice-cold phosphate-buffered saline (PBS) (pH 7.4) containing 1% Triton X-100, 2 mM EDTA, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium vanadate, 10 mM sodium fluoride, and proteinase inhibitors cocktail (Sigma-Aldrich). Lysates were centrifuged at 15,000 rpm for 10 min at 4C, and protein concentration was measured using the Bradford protein assay (Bio-Rad). One milligram of lysates was incubated with 2 g of anti-Myc-tag antibody (catalog no. 16286-1-AP, Proteintech, Rosemont, IL, USA) in the presence of Protein A/G Agarose (Santa Cruz Biotechnology) at 4C overnight. After washing twice with ice-cold modified PBS buffer (pH 7.4) (287 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Triton X-100, and 1 mM EDTA), resins were incubated in 30 l of 0.2 M glycine-HCl buffer (pH 2.5) (Polysciences Inc. Warrington, PA, USA) at RT for 15 min, and then the supernatants were collected and neutralized by adding an equal amount of 1 M tris-HCl (pH 8.0) (Sigma-Aldrich). To increase stringency during the washing, NaCl concentration was increased from 137 to 287 mM in the modified PBS buffer to reduce ionic protein interaction. Eluted immunoprecipitates or whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for Myc-tag (dilution 1:5000) (catalog no. 2276, Cell Signaling Technology), FLAG-tag (1:25,000) (catalog no. F1804, Sigma-Aldrich), and HA-tag (1:3000) (catalog no. sc-7392, Santa Cruz Biotechnology). Anti-mouse antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific) was used as a secondary antibody (dilution 1:25,000). Enhanced chemiluminescence reagent, Lumigen ECL Ultra (TMA-6) (Lumigen, Southfield, MI, USA), was used to detect the antigen-antibody complexes.

To assess the functional activity of AAV-cTuberin-Myc, we cotransfected HEK293T cells, as previously described with minor modifications (60). Plasmids included HA-tagged p70S6 kinase (HA-p70S6K) (60), which is phosphorylated (pS6K T389) by mTORC1 and was used as a reporter for mTORC1 activation, and Flag-tagged hamartin (Flag-hamartin) (60), along with AAV-cTuberin-Myc. Full-length Flag-tagged tuberin (Flag-tuberin) (60) was used as a positive control, and AAV-GFP was used as a negative control. Transfections were carried out for 48 hours using Lipofectamine 2000. Cell lysates were prepared using radioimmunoprecipitation assay lysis buffer, and immunoblotting was performed, as described (60). Briefly, proteins were separated on a Novex 4 to 12% tris-glycine gradient gel (Life Technologies) followed by transfer to 0.45 M nitrocellulose membrane (Bio-Rad). Antibodies included M2 anti-Flag mouse monoclonal (Sigma-Aldrich), anti-hamartin and anti-pS6K (T389) (Cell Signaling Technology), anti-Myc mouse monoclonal (9E10, University of Iowa Hybridoma Bank), and anti-HA mouse monoclonal (HA.11, BioLegend/Covance, San Diego, CA, USA).

HEK293T cells were seeded in a six-well plate (500,000 cells per well) for 24 hours. The cells were then transfected with plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 2.5 g/500,000 cells using Lipofectamine 2000 in Opti-MEM. Six hours later, transfection media was removed and replaced with DMEM (10% FBS and 1% PS), and cells were grown for 72 hours. Cells were washed twice in PBS, and proteins were extracted with protein extraction solution (PRO-PREP, iNtRON Biotechnology, Korea) for 20 min at 20C. The cell lysates were centrifuged at 14,000g at 4C. Protein concentrations of cell lysates were determined using a Bio-Rad protein assay kit. Equal amounts of protein (20 g) were separated using 4 to 12% precast NuPAGE bis-tris SDS-PAGE gels (Invitrogen) and transferred onto nitrocellulose membranes (Thermo Fisher Scientific Inc., Rockford, IL, USA). Membranes were blocked for 1 hour in tris-buffered saline (TBS) with 0.1% Tween 20 and 5% nonfat dry milk, followed by an overnight incubation with primary antibody to tuberin (#3990, 1:1000 dilution, Cell Signaling Technology diluted in the same buffer at 4C). On the next day, the membranes were washed with TBS with 0.1% Tween 20 (three times, 5 min each) followed by incubation with the appropriate horseradish peroxidaseconjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at RT. An enhanced chemiluminescence kit (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific, Waltham, MA, USA) was used to detect protein expression. The optical density of each band was determined on Western blots scanned with a G:Box (Syngene, Cambridge, UK).

Brains and livers were flash-frozen to determine AAV genome biodistribution and expression of transgene mRNA. Genomic and AAV vector DNA was isolated using the Qiagen DNeasy Blood and Tissue Kit (catalog no. 69504) according to the manufacturers instruction. Total RNA was extracted using the Qiagen RNeasy Lipid Tissue Mini Kit (catalog no.74804) and Qiagen RNeasy Mini Kit (catalog no. 74104), with additional on-column deoxyribonuclease (DNase) digestion with the Qiagen RNase-free DNase set (catalog no. 79254) to ensure digestion of AAV-cTuberin genomes. Then, extracted RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Master Mix (Thermo Fisher Scientific, catalog no. 11754-050), according to the manufacturers protocol. A no-RT set of samples for the AAV-cTuberin group was included to confirm detection of cDNA derived from cTuberin mRNA and not contaminating AAV-cTuberin genomes. Using 50-ng genomic DNA as template, TaqMan qPCR was performed using custom TaqMan probe and primers to 3 end of cTuberin and c-Myc tag of the transgene expression cassette (forward primer, 5-AGCCAACACCAGGATACGAA-3; reverse primer, 5-GCTAATCAGCTTCTGCTCCAC-3; probe, 5-FAM- AGCGGCTGATCTCCTCCGTGG-MGB-3) (fig. S5). For each sample, a separate qPCR was performed using TaqMan probe and primer sets (Thermo Fisher Scientific, assay ID Mm01180221_g1, gene symbol Gm12070) that detects GAPDH genomic DNA, to ensure equal genomic DNA input for each sample. For each organ/tissue, the AAV vector genome copies for each sample were adjusted by taking into account any differences in GAPDH Ct values using the following formula: (AAV vector genome copies)/(2Ct). The Ct value was calculated as GAPDH Ct value (sample of interest) average GAPDH Ct value (sample with highest Ct value). Data were expressed as AAV vector genomes per 50 ng of genomic DNA.

Experimental research protocols were approved by the Institutional Animal Care and Use Committee for the Massachusetts General Hospital (MGH) following the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. Experiments were performed on Tsc2c/c-floxed mice [Tsc2-floxed; (61)]. These mice have a normal, healthy life span. In response to Cre recombinase, the Tsc2c/c alleles are converted to null alleles. For vector injections, in the neonatal period (P0 to P3), pups were cryo-anesthetized and injected with 1 to 2 l of viral vector AAV1-CBA-Cre into each cerebral lateral ventricle with a glass micropipette (70 to 100 mm in diameter at the tip) using a Narishige IM300 microinjector at a rate of 2.4 psi/s (Narshige International, East Meadow, NY, USA). Mice were then placed on a warming pad and returned to their mothers after regaining normal color and full activity typical of newborn mice. At 3 weeks of age (P21), mice were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL, USA) inhalation [3.5% isoflurane in an induction chamber and then maintained anesthetized with 2 to 3% isoflurane and oxygen (1 to 2 liters/min) for the duration of the injection]. AAV vectors were injected retro-orbitally into the vasculature in a volume of 60 l (AAV1 or AAV9) of AAV-cTuberin-Myc using a 0.3-ml insulin syringe over less than 2 min (62) or noninjected.

Eighteen measurements of the body weight of the animals were recorded from P23 to P50. To assess motor coordination, animals were placed on an automated rotarod apparatus (Harvard Apparatus, Holliston, MA, USA) using accelerated velocities (4 to 64 rpm over 120 s). Each animal was assessed three times with 5-min rest intervals in each session for nine sessions 3 to 4 days apart. For each assessment, the time ended when the mouse fell off the treadmill or when the time interval elapsed. All functional assessment tests were performed blinded with respect to the mouse genotype.

HEK293T cells were seeded on coverslip coated with poly-d-lysine (25,000 cells per coverslip) for 24 hours. The cells were then transfected with plasmid DNAs (AAV-null, AAV-GFP, and AAV-cTuberin) at 250 ng/25,000 cells using Lipofectamine 2000 in Opti-MEM. Six hours later, transfection media was removed and replaced with DMEM (10% FBS and 1% PS), and cells were grown for 72 hours. The cells were fixed with 4% paraformaldehyde (PFA) (Boston BioProducts, Ashland, MA, USA) for 10 min at RT followed by permeabilization using 0.01% Triton X-100 (Sigma-Aldrich) in PBS (PBST) for 10 min at RT. The cells were then blocked with 3% bovine serum albumin (BSA) in PBST for 1 hour at RT, followed by overnight incubation with primary antibodies at 4C [primary antibodies: c-Myc (1:400 dilution; 9E10, Life Technologies)] and GFP (1:400 dilution; A11122, Life Technologies). The cells were then washed three times for 5 min in PBST and incubated with secondary antibody (goat anti-mouse 488, Jackson ImmunoResearch Laboratories) (1:400 dilution), for 1 hour at RT. The cells were washed three times for 5 min using PBST, mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA). Note that, unfortunately, we were not able to detect cMyc in brain sections using several sources of c-Myc antibodies.

The mouse brains were harvested and subjected for standard histological processing as described (14). Five-micrometer sections were stained with hematoxylin and eosin. For frozen sections, adult mice were euthanized using ketamine/xylazine (100:10) (Akorn Inc., Lake Forest, IL, USA) followed by transcardiac perfusion with 1 PBS and 4% PFA in PBS overnight at 4C, cryo-protected with 25% sucrose in PBS, and embedded in optimal cutting temperature medium (catalog no. 4583, Tissue Teck). Brain sections were prepared in 10-mm coronal sections and were blocked in 10% BSA in 1 PBS + 0.3% Triton X-100 for 1 hour at RT and subsequently incubated with rabbit anti-Ki67 (1:1000; #ab15580, Abcam) or rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:400; #2211, Cell Signaling Technology) overnight at 4C. Following three washes in 0.1 PBS, the sections were incubated with secondary antibody Alexa 555 (1:400; Jackson ImmunoResearch Laboratories) for 1 hour at RT. The sections were then washed three times with 1 PBS and mounted with DAPI mounting medium (Vectashield, #H-1200).

Whole mouse brain sections immunostained for pS6 (biological triplicates for each group, three coronal sections per mouse) were imaged using a Nikon Ti2 inverted microscope equipped with W1 Yokogawa Spinning disk scanhead with 50-m pinholes, a Toptica 4 laser launch, and an Andor Zyla 4.2 Plus sCMOS monochrome camera. The slides were mounted on a Nikon linear encoded motorized stage, and the mouse whole brain sections were scanned using Plan Apo 20/0.8 differential interference contrast (DIC) I objective lens objective lens at 405 nm for DAPI (100-ms exposure) and 561 nm for pS6 staining (100-ms exposure). Signals were collected using a Semrock di01-t405/488/568/647 dichroic mirror and Chroma 455/50 or 605/52 nm emission filters. Images were captured using NIS AR 5.02 acquisition software and 12-bit gain four-camera setting. A series of images were captured and stitched together using blending algorithm with 15% overlap among images.

Stitched images were analyzed in Fiji, an open source image processing package based on ImageJ (63). All images were thresholded within the 80 to 800 tonal range for both DAPI and pS6 staining. An outline was manually drawn to delineate choroid plexuses, ventricles, large empty spots, and meninges from the whole mouse brain section image. These regions are known to contain significant amounts of autofluorescence and therefore were excluded from downstream analysis. Within the confined region of interests (ROIs), we measured the area for the whole brain section. To identify pS6 puncta size and intensity within them, the thresholded pS6 channel image was converted into eight-bit image and further thresholded within the 70 to 255 tonal range. Subsequently, particle analysis was performed to identify any puncta within 5 to 200 m2 and 0.1 to 1.0 circularity parameters. The area for each punctum was measured. These puncta ROIs were then used to identify raw integrated density on original unthresholded 12-bit brain section images. Normalized pS6 puncta number of a brain section was calculated by dividing the total number of pS6 puncta by the brain section area.

All analyses of survival curves (Mantel-Cox test and log-rank test) were performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Flow cytometry analysis on c-Mycpositive cells was analyzed using unpaired t test. Western blot analysis on pS6 and tuberin expression levels in the mouse brain and PS6 puncta parameters were analyzed using unpaired t test. LDH cytotoxicity assay and Western blot analysis on relative levels of S6K T389 phosphorylation were analyzed using one-way analysis of variance (ANOVA) test. P values of <0.05 were considered statistically significant.

Acknowledgments: We thank S. McDavitt for editorial assistance, M. F. Lee (Medical Photographer in Pathology Media Laboratory, MGH) for imaging training, M. Zinter (Vector Core, MGH, Charlestown, MA, USA) for AAV vector packaging, and M. Whalen for the use of the rotarod. Funding: This work was supported by DOD Army Grant W81XWH-13-1-0076 (to X.O.B.), NIH R01GM115552 (to M.K.), NIH NIDCD R01DC017117-01A1 (to C.A.M.), NIH NINDS 1R61NS108232 (to X.O.B., C.A.M., and V.R.), and NIH NS109540 (to V.R.). We would like to acknowledge the MGH Vector Core for the production of viral vectors (supported by NIH/NINDS P30NS045776; B.A.T.) and P. M. Llopis, Microscopy Resources on the North Quad (MicRoN), Harvard Medical School, NRB-Longwood, MA, USA. Author contributions: X.O.B., S.P., D.Y., C.A.M., and M.K. conceived and designed the experiments. S.P., P.-S.C., R.L.B., X.Z., and S.K. performed the experiments. S.P., P.-S.C., K.-H.L., and S.K. analyzed the data. S.P., P.-S.C., D.Y., B.A.T., E.A.T., X.Z., R.L.B., R.T.B., D.J.K., A.S.-R., B.G., K.-H.L., V.R., M.K., C.A.M., and X.O.B. wrote and edited the paper. Competing interests: X.O.B., S.P., D.Y., and C.A.M. have filed a provisional patent application for the cTuberin construct. C.A.M. has a financial interest in Chameleon Biosciences Inc., a company developing an enveloped AAV vector platform technology for repeated dosing of systemic gene therapy. X.O.B., V.R., and C.A.M.s interests are reviewed and managed by MGH and Partners HealthCare in accordance with their competing interest policies. All other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Plasmid requests can be provided by MGH pending scientific review and a completed material transfer agreement. Requests for the plasmid should be submitted to C.A.M. at cmaguire{at}mgh.harvard.edu.

See the article here:
Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin - Science Advances

BEIJING, Jan. 8 (Xinhua) -- For the first time, a genome-wide CRISPR-based screening technology has identified a new driver of cellular senescence. It can form part of new strategies to delay aging and prevent aging-associated diseases, Chinese researchers said.

By screening and identifying more than 100 genes responsible for the aging of human cells, the research team demonstrated that knocking out, or disabling, some genes by CRISPR can discourage the aging of human mesenchymal precursor cells (hMPCs). Among the genes that lead to senility, and KAT7 (a histone acetyltransferase), is one of the catalysts for aging.

Knocking out KAT7 has been proven effective in alleviating cellular senescence in the team's experiments, said Zhang Weiqi, a researcher at the Beijing Institute of Genomics under the Chinese Academy of Sciences. The scientists managed to reduce the proportion of the senescent cells in the livers of aged mice and prolonged the lifespan of physiologically aged mice and those with progeria.

The novel gene therapy, based on disabling a single gene or using KAT7 inhibitors, could extend mammal life. It could also slow down the aging of human liver cells. It suggests a massive potential for its application in translational medicine against human aging.

The study was published on Thursday in Science Translational Medicine online.

Originally posted here:
Chinese researchers discover new anti-aging gene therapy - The Star Online

Sana Biotechnology was founded in 2018 with a mission of solving some of the most difficult challenges in gene and cell therapy. Toward that end, the company is engineering hypoimmune stem cells that can evade detection and destruction by the immune system.

Now, some of Sanas founders, who are scientists at the University of California, San Francisco (UCSF), are describing how these engineered stem cells are able to shut down the immune systems natural killer (NK) cells. They believe their findings could enhance the development of implantable cell therapies, as well as cancer immunotherapies, they reported in the Journal of Experimental Medicine.

The ability to evade NK cells could enhance a range of experimental treatments, including implants of insulin-producing cells for patients with diabetes and cardiac cells to repair heart damage. These cells are typically rejected by the immune systema problem hypoimmune stem cells were designed to circumvent.

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The UCSF team used gene modification technology to design the cells so they avoid the immune responses that are either built into the bodys defense system or learned. The researchers achieved that feat by engineering the cells to express the protein CD47, which shuts down innate immune cells by activating signal regulatory protein alpha, or SIRP-alpha.

The researchers were surprised to discover that the hypoimmune stem cells were able to escape NK cells, even though NK cells were not previously known to express SIRP-alpha. Rather than studying lab-grown cell lines, they took cells directly from patients. Thats where they found SIRP-alpha.

Whats more, the UCSF team discovered that NK cells begin to express SIRP-alpha after they are activated by cytokines that are typically abundant in inflammatory states.

RELATED: Fierce Biotech's 2020 Fierce 15 | Sana Biotechnology

To further prove out the utility of engineered stem cells, the UCSF researchers implanted cells with rhesus macaque CD47 into monkeys. They documented the activation of SIRP-alpha in NK cells. Those NK cells did not kill the transplanted cells.

A similar technique could be used, but in reverse, to implant pig cardiac cells into people, the UCSF team argued. If human CD47 were engineered into pig heart cells, they could be implanted into people without risking rejection by NK cells, they suggested.

Sana made waves in 2018 when it raised a whopping $700 million in a single venture round from the likes of Arch Venture Partners, Flagship Pioneering and Bezos Expeditions. We believe that one of, if not the most, important thing happening in medicine over the next several decades is the ability to modulate genes, use cells as medicines, and engineer cells, said Steve Harr, president and CEO of Sana, at the time.

Sana did not provide materials or funding for the new study, but it is now developing the hypoimmune stem cell technology for clinical testing.

The UCSF team believes their findings could also boost cancer immunotherapy. The engineered cells could help combat checkpoints that allow tumors to evade immune detection, they said.

"Many tumors have low levels of self-identifying MHC-I protein and some compensate by overexpressing CD47 to keep immune cells at bay," said Lewis Lanier, Ph.D., director of the Parker Institute for Cancer Immunotherapy at the UCSF Helen Diller Family Comprehensive Cancer Center, in a statement. "This might be the sweet spot for antibody therapies that target CD47."

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Engineered stem cells that evade immune detection could boost cell therapy and I-O - FierceBiotech

At the end of the study, it was found that the rats had regained their ability to use their paws and were able to pick up sugar cubes to feed themselves, according to The Independent. The gene therapy trial was conducted at Kings College London, U.K. The focus of the work was to repair damage to the spinal cords of the rodents. The spinal cords of the rats had been purposefully damaged to mimic the damaged sometimes suffered to humans after car crashes. Quoted by Sky News, Professor Elizabeth Bradbury, one of the principal researchers, stated: "In some of the tests we looked at such as gripping the rungs of a ladder the treatment worked within one to two weeks."Gene therapyGene therapy is an important aspects of medicine. The process is designed to introduce genetic material into cells. This is to compensate for abnormal genes or, alternatively, to produce a beneficial protein. In cases where a mutated gene causes a necessary protein to be faulty or to become missing, then gene therapy could work to introduce a normal copy of the gene and hence to restore the function of the protein.There are different variants of gene therapy, including plasmid DNA, where circular DNA molecules are genetically engineered so they carry therapeutic genes into human cells; viral vectors, where viruses are used to deliver genetic material into cells; bacterial vectors, where bacteria are modified and then deployed as vehicles to carry therapeutic genes into human tissues; and human gene editing technology, where genes are edited to disrupt harmful genes or to repair mutated genes. There is also patient-derived cellular gene therapy products. With this more recent process, cells are taken from the patient, modified and then returned to the patient.For some scientists, the next phase is germinal gene therapy. This has been achieved experimentally in animals but not in humans.Novel researchWith the new study, the process involved injecting a gene that produces an enzyme called chondroitinase, into the spinal cords of the rats. This enzyme functions to breaks down scar tissue, a tissue that is formed following damage to the spinal cord. he tissue prevents new connections from being formed between nerves. The enzyme is also being used in trials for vitreous attachment and for treating cancer.

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article image Advances in gene therapy to help paralysis - Digital Journal

SAN DIEGO and STOCKHOLM, Sweden, Jan. 07, 2021 (GLOBE NEWSWIRE) -- Oncternal Therapeutics, Inc. (Nasdaq: ONCT), a clinical-stage biopharmaceutical company focused on the development of novel oncology therapies, today announced that it established a research and development collaboration with world-renowned Karolinska Institutet in Stockholm, Sweden, to advance novel ROR1-targeting cell therapies focused on CAR-T cells and CAR-NK (Natural Killer) cells from the laboratory into the clinic.

As part of the collaboration, IND-supporting preclinical studies will be performed in the Cell and Gene Therapy Group led by Evren Alici, M.D. Ph.D., within the NextGenNK Center, which is a Competence Center for the development of next-generation NK cell-based cancer immunotherapies. The Center is coordinated by Karolinska Institutet and collaborates with the Karolinska University Hospital as well as prominent national and international industrial partners. The Center was launched in 2020, and is jointly funded by Swedens innovation agency Vinnova, Karolinska Institutet, and the industrial partners.

Given that NK cells were discovered at Karolinska Institutet, we are excited to work together with industry partners to translate scientific advances into next-generation cell therapies that will benefit cancer patients, said Hans-Gustaf Ljunggren, M.D. Ph.D., Director of the NextGenNK competence center. We look forward to collaborating with the outstanding team at Oncternal to develop cutting-edge T and NK cell therapies targeting ROR1, which is a promising target in many oncology indications. It could be ideally suited for cell therapy.

We are honored to work together with the world-leading academic team at Karolinska Institutet to accelerate the development of our ROR1-targeting CAR-T cell immunotherapy program, said James Breitmeyer, M.D., Ph.D., Oncternals President and CEO. ROR1 has emerged as an important and underexplored target for cancer therapy, and we believe that ROR1-targeting CAR-T and CAR-NK therapies hold significant promise for patients with both hematologic cancers and solid tumors. We believe that utilizing the ROR1 binding domain of our clinical-stage antibody cirmtuzumab as a component of the CAR has the potential to give us a safety and efficacy advantage.

About Oncternal TherapeuticsOncternal Therapeutics is a clinical-stage biopharmaceutical company focused on the development of novel oncology therapies for the treatment of cancers with critical unmet medical need. Oncternal focuses drug development on promising yet untapped biological pathways implicated in cancer generation or progression. The clinical pipeline includes cirmtuzumab, an investigational monoclonal antibody designed to inhibit the ROR1 (Receptor-tyrosine kinase-like Orphan Receptor 1) pathway, a type I tyrosine kinase-like orphan receptor, that is being evaluated in a Phase 1/2 clinical trial in combination with ibrutinib for the treatment of patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) and in an investigator-sponsored, Phase 1b clinical trial in combination with paclitaxel for the treatment of women with HER2-negative metastatic or locally advanced, unresectable breast cancer. The clinical pipeline also includes TK216,an investigational targeted small-molecule inhibitor of the ETS family of oncoproteins, that is being evaluated in a Phase 1 clinical trial for patients with Ewing sarcoma alone and in combination with vincristine chemotherapy. In addition, Oncternal has a program utilizing the cirmtuzumab antibody backbone to develop a CAR-T therapy that targets ROR1, which is currently in preclinical development as a potential treatment for hematologic cancers and solid tumors. More information is available at http://www.oncternal.com.

About KarolinskaInstitutetKarolinska Institutetis one of the worlds leading medical universities. Its vision is to advance knowledge about life and strive towards better health for all. Karolinska Institutet accounts for the single largest share of all academic medical research conducted in Sweden and offers the countrys broadest range of education in medicine and health sciences. The Nobel Assembly at Karolinska Institutet selects the Nobel laureates in Physiology or Medicine.

Forward-Looking InformationOncternal cautions you that statements included in this press release that are not a description of historical facts are forward-looking statements. In some cases, you can identify forward-looking statements by terms such as may, will, should, expect, plan, anticipate, could, intend, target, project, contemplates, believes, estimates, predicts, potential or continue or the negatives of these terms or other similar expressions. These statements are based on the companys current beliefs and expectations. Forward looking statements include statements regarding Oncternals beliefs, goals, intentions and expectations including, without limitation, Oncternals belief that ROR1-targeting CAR-T and CAR-NK therapies hold significant promise for patients with hematologic cancers and solid tumors; whether using ROR1 binding domain as a component of the CAR therapeutic candidate will provide a safety or activity advantage over other drugs or drug candidates; the potential that ROR1 could be an ideal target for cell therapy; and other statements regarding Oncternals development plans. Forward looking statements are subject to risks and uncertainties inherent in Oncternals business, which include, but are not limited to: the risk that the collaboration with Karolinska Institutet will not generate any intellectual property or otherwise identify drug candidates for development or provide Oncternal any benefits; the COVID-19 pandemic may disrupt Oncternals business operations or the business operations of Karolinska Institutet, increasing their respective costs; uncertainties associated with the clinical development and process for obtaining regulatory approval of product candidates, including potential delays in the commencement, enrollment and completion of clinical trials; Oncternals dependence on the success of cirmtuzumab, TK216 and its other product development programs; the risk that competitors may develop technologies or product candidates more rapidly than Oncternal, or that are more effective than Oncternals product candidates, which could significantly jeopardize Oncternals ability to develop and successfully commercialize its product candidates; Oncternals limited operating history and the fact that it has incurred significant losses, and expects to continue to incur significant losses for the foreseeable future; the risk that the company will have insufficient funds to finance its planned operations and may not be able to obtain sufficient additional financing when needed or at all as required to achieve its goals, which could force the company to delay, limit, reduce or terminate its product development programs or other operations; and other risks described in the companys prior press releases as well as in public periodic filings with the U.S. Securities & Exchange Commission. All forward-looking statements in this press release are current only as of the date hereof and, except as required by applicable law, Oncternal undertakes no obligation to revise or update any forward-looking statement, or to make any other forward-looking statements, whether as a result of new information, future events or otherwise. All forward-looking statements are qualified in their entirety by this cautionary statement. This caution is made under the safe harbor provisions of the Private Securities Litigation Reform Act of 1995.

Oncternal Contacts:

Company ContactRichard Vincent 858-434-1113rvincent@oncternal.com

Investor ContactCorey Davis, Ph.D. LifeSci Advisors 212-915-2577 cdavis@lifesciadvisors.com

Source: Oncternal Therapeutics, Inc.

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Oncternal Therapeutics and Karolinska Institutet Establish Collaboration for Research and Development of ROR1-targeting CAR-T and CAR-NK Cell...

SAN FRANCISCO--(BUSINESS WIRE)--AllStripes (formerly RDMD), a healthcare technology company dedicated to accelerating research for patients with rare diseases, today announced a multiyear collaboration with Taysha Gene Therapies, Inc. (NASDAQ: TSHA), a patient-centric gene therapy company focused on developing and commercializing AAV-based gene therapies for the treatment of monogenic diseases of the central nervous system in both rare and large patient populations.

The collaboration will focus on advancing the development of TSHA-104, an AAV9-based gene therapy in development for SURF1-associated Leigh syndrome, a deadly rare disease that primarily affects infants. AllStripes will use its platform, which gives patients control over their health histories, to unify otherwise scattered and fragmented SURF1-associated clinical data, allowing researchers to uncover new insights into the natural history and burden of disease and better inform the development of clinical studies.

This collaboration will allow us to leverage the AllStripes technology platform to optimize our therapeutic strategy and to potentially accelerate the development of TSHA-104 in SURF1-associated Leigh syndrome, said RA Session, II, president, founder and chief executive officer of Taysha. We remain committed to developing a safe and effective gene therapy for patients suffering with this devastating disease, and data generated from this unique collaboration could bring us one step closer to our goal.

Mutations in the SURF1 gene prevent mitochondria from producing enough energy for cells in the body to function normally, leading to Leigh syndrome, a severe and rare neurological disorder characterized by progressive loss of mental and movement abilities. SURF1-associated Leigh syndrome typically presents during infancy or early childhood, and often results in death within a few years. Approximately 10-15% of people with Leigh syndrome have a SURF1 mutation. There is currently no targeted treatment or cure for SURF1-associated Leigh syndrome.

Taysha has brought together accomplished and knowledgeable gene therapy and CNS disease experts to develop potentially transformative therapies, said Nancy Yu, co-founder and chief executive officer of AllStripes. With no available treatment for SURF1-associated Leigh syndrome, we are very pleased to empower patients and their families with an avenue to participate in research that will support the development path of TSHA-104. We are hopeful that this novel gene therapy will bring meaningful benefit to children and their families, and give them more time together.

TSHA-104 has been granted rare pediatric disease and orphan drug designations from the U.S. Food and Drug Administration (FDA) for the treatment of SURF1-associated Leigh syndrome. An Investigational New Drug (IND) application for TSHA-104 in SURF1-associated Leigh syndrome is expected to be submitted to the FDA in 2021.

About Taysha Gene Therapies

Taysha Gene Therapies (Nasdaq: TSHA) is on a mission to eradicate monogenic CNS disease. With a singular focus on developing curative medicines, we aim to rapidly translate our treatments from bench to bedside. We have combined our teams proven experience in gene therapy drug development and commercialization with the world-class UT Southwestern Gene Therapy Program to build an extensive, AAV gene therapy pipeline focused on both rare and large-market indications. Together, we leverage our fully integrated platforman engine for potential new cureswith a goal of dramatically improving patients lives. More information is available at http://www.tayshagtx.com.

About AllStripes

AllStripes is a healthcare technology company dedicated to unlocking new treatments for people with rare diseases. AllStripes has developed a technology platform that generates FDA-ready evidence to accelerate rare disease research and drug development, as well as a patient application that empowers patients and families to securely participate in treatment research online and benefit from their own medical data. AllStripes was founded by CEO Nancy Yu and technology developer Onno Faber, following his diagnosis and journey with the rare disease neurofibromatosis type 2. The company is backed by Lux Capital, Spark Capital, Maveron Capital, Village Global, Garuda Ventures and a number of angel investors. For more information, visit http://www.allstripes.com.

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AllStripes Announces Collaboration with Taysha Gene Therapies for SURF1-Associated Leigh Syndrome Program - Business Wire

OTTAWA, Jan. 08, 2021 (GLOBE NEWSWIRE) -- The global regenerative medicine market is representing impressive CAGR of 16.1% during the forecast period 2020 to 2027.

Regenerative medicine is the division of medicine that promotes methods to repair, regrow or replace injured or diseased tissues, organs or cells. Regenerative medicine comprises of the formation and use of remedial stem cells, manufacturing of artificial organs, and tissue engineering. The combinations of tissue engineering, cell and gene therapies can strengthen the natural healing procedure in the places it is desired most, or occupy the role of a permanently injured organ. Regenerative medicine is a rather new field that connects experts in chemistry, biology, engineering, computer science, robotics, medicine, genetics and other domains to find explanations to some of the most interesting medical problems confronted by humankind.

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Growth Factors:

Factors such as increasing prevalence of chronic disorders and genetic disorders, increasing popularity of stem cells, increasing number of trauma emergencies is driving the growth of regenerative medicine market. An illness or disorder that usually persists for 3 months or longer and might get worse over a period is termed as chronic disorder. Chronic diseases mostly occur in the elderly people and can typically be controlled but not repaired. The most prevalent types of chronic ailments are heart disease, arthritis, cancer, diabetes, and stroke. Cardiovascular disorders are the biggest cause of deaths worldwide. As per the WHO data, deaths due to cardiovascular disorders represent almost 31% of the deaths globally. Almost 85% of these demises are due to stroke and heart attack. Diabetes is another most prevalent chronic ailment that affects millions of people globally. According to International Diabetes Federation (IDF), around 463 million adults (age group: 20-79 years) are battling with diabetes and by the year 2045 the number will rise to a staggering 700 million. Furthermore, approximately 75% of all health care expenses are owed to chronic ailments. Four out of the five most costly health conditions are chronic disorders such as cancer, heart disease, pulmonary conditions, and mental disorders. Regenerative medicine approaches such as stem cell therapy can cure the chronic ailments such as diabetes and arthritis, which otherwise require lifetime of medications.

The role of regenerative medicine in post trauma recovery is constantly evolving as more and more research is showing positive results. The use of regenerative medicine can be a landmark moment in the history of healthcare that will transform the treatment of chronic ailments and trauma related conditions. Thus, the high incidence of chronic ailments is driving the growth of regenerative medicine market.

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Regional Analysis:

The report covers data for North America, Europe, Asia Pacific, Latin America, and Middle East and Africa. In 2019, North America dominated the global market with a market share of more than 45%. U.S. represented the highest share in the North American region primarily due to constant activity in the field of drug discovery and tissue engineering. Moreover, early adoption of latest healthcare technologies also contributed to the high market share of the United States.

Europe was the second important market chiefly due to favorable reimbursement scenario and presence of latest healthcare infrastructure. The presence of skilled researchers in the European region is also expected to boost the demand for regenerative medicine market in the near future. Asia Pacific is anticipated to grow at the maximum CAGR of around18% in the forecast period due to high incidence of trauma cases and chronic disorders. Latin America and the African and Middle Eastern region will display noticeable growth.

Report Highlights:

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Key Market Players and Strategies:

The major companies operating in the worldwide regenerative medicine are Integra Life Sciences Corporation, Aspect Biosystems, Amgen, Inc., Medtronic plc, AstraZeneca, Novartis AG, Smith & Nephew plc, MiMedx Group, Shenzhen SibionoGeneTech Co., Ltd., and Baxteramong others.

High investment in the research and development along with acquisition, mergers, and collaborations are the key strategies undertaken by companies operating in the global regenerative medicine market. Recently Fuse Medical, Inc., an evolving manufacturer and supplier of innovative medical devices for the spine and orthopedic marketplace, declared the launch of FuseChoice Plus and FuseChoice Umbilical and Amniotic Membranes, and FuseChoice Plus Amniotic Joint Cushioning Fluid, the newest additions to a wide-ranging line of biologics product offerings.

Market Segmentation

By Product

By Application

By Geography

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Precedence Research is a worldwide market research and consulting organization. We give unmatched nature of offering to our customers present all around the globe across industry verticals. Precedence Research has expertise in giving deep-dive market insight along with market intelligence to our customers spread crosswise over various undertakings. We are obliged to serve our different client base present over the enterprises of medicinal services, healthcare, innovation, next-gen technologies, semi-conductors, chemicals, automotive, and aerospace & defense, among different ventures present globally.

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Regenerative Medicine Market to Reach Valuation US$ 23.7 Bn by 2027 - GlobeNewswire

Dublin, Jan. 06, 2021 (GLOBE NEWSWIRE) -- The "Cell and Gene Therapy Global Market Report 2020-30: COVID-19 Growth and Change" report has been added to ResearchAndMarkets.com's offering.

Cell and Gene Therapy Global Market Report 2020-30: COVID-19 Growth and Change provides the strategists, marketers and senior management with the critical information they need to assess the global cell & gene therapy market.

Major players in the cell and gene therapy market are Gilead Sciences, Bristol-Myers Squibb, Novartis AG, Amgen, Merck, Organogenesis Holdings, Dendreon, Vericel, Bluebird Bio and Fibrocell Science.

The global cell and gene therapy market is expected to decline from $6.68 billion in 2019 to $6.92 billion in 2020 at a compound annual growth rate (CAGR) of 3.61%. The slow growth is mainly due to the COVID-19 outbreak that has led to restrictive containment measures involving social distancing, remote working, and the closure of industries and other commercial activities resulting in operational challenges. The entire supply chain has been disrupted, impacting the market negatively. The market is then expected to recover and reach $13.23 billion in 2023 at a CAGR of 24.10%.

The cell and gene therapy market consists of sales of cell and gene therapies by entities (organizations, sole traders and partnerships) that develop cell and gene therapies. Cell therapy refers to the transfer of intact, live cells that are originated from autologous or allogenic sources and gene therapy refers to the introduction, removal, or change in the genome for treating diseases. The market consists of revenue generated by the companies developing cell and gene therapy products by the sales of these products.

North America was the largest region in the cell and gene therapy market in 2019. It is also expected to be the fastest-growing region in the forecast period.

In December 2019, Roche, a Swiss multinational healthcare company, acquired Spark Therapeutics for $4.3 billion. The acquisition supports the commitment of Roche to bring transformational therapies and innovative approaches to people with serious illnesses. Spark Therapeutics will continue to work within the Roche Group as an independent company. Spark Therapeutics, headquartered in Philadelphia, is a fully integrated commercial company involved in the discovery, production, and distribution of gene therapies for genetic disorders including blindness, hemophilia, lysosomal storage, and neurodegenerative diseases.

The cell and gene therapy market covered in this report is segmented by product into cell therapy; gene therapy and by application into oncology; dermatology; musculoskeletal; others.

Limited reimbursements preventing patients from receiving treatments are expected to limit the growth of cell and gene therapy (CGT market. In 2019, Trinity Life Sciences, a life sciences solution provider, researched national and large regional commercial health insurance plans in the US. It found that the confluence of increasing price, patient volume and number of CGTs on the market is likely to change the reimbursement model for CGTs and impact payer budgets by 5-10%. Payers realize that financing needs to be generated for cost management due to the uncertainty surrounding reimbursement of ancillary costs. Limited reimbursements and uncertain insurance plans are preventing patients from receiving high-cost CGT, which is expected to limit market growth.

Chimeric antigen receptor (CAR) T-cell therapy is shaping the cell and gene therapy (CGT) market. (CAR) T-cell therapy is a combination of cell and gene therapy in which T cells are collected from the patient's blood and are genetically engineered to produce modified receptors at their surface, known as chimeric antigen receptors (CARs). These modified T cells with special structures (receptors) are reinfused into the patient. Then, the modified receptors of T cell help in targeting the surface antigen of the cancer cell that ultimately results in the killing of tumor cells in patients.

In 2020, the US-FDA approved Bristol-Myers Squibb's two CAR-T cell therapies to treat lymphoma and multiple myeloma and is set to be launched. Currently, FDA approved CAR-T cell therapy treatments like Tisagenlecleucel for the treatment of B-cell precursor acute lymphoblastic leukemia (ALL) in children and Axicabtagene ciloleucel for the treatment of adult patients with relapsed or refractory large B-cell lymphoma.

Steady investment and consolidation in cell and gene therapies contributed to the growth of the cell and gene therapy (CGT) market. After recognizing the potential of the CGT market, 16 out of the 20 largest biopharma companies by revenue, added CGT products to their portfolio.

Key Topics Covered:

1. Executive Summary

2. Cell And Gene Therapy Market Characteristics

3. Cell And Gene Therapy Market Size And Growth 3.1. Global Cell And Gene Therapy Historic Market, 2015 - 2019, $ Billion 3.1.1. Drivers Of The Market 3.1.2. Restraints On The Market 3.2. Global Cell And Gene Therapy Forecast Market, 2019 - 2023F, 2025F, 2030F, $ Billion 3.2.1. Drivers Of The Market 3.2.2. Restraints On the Market

4. Cell And Gene Therapy Market Segmentation 4.1. Global Cell And Gene Therapy Market, Segmentation By Product, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

4.2. Global Cell And Gene Therapy Market, Segmentation By Application, Historic and Forecast, 2015-2019, 2023F, 2025F, 2030F, $ Billion

5. Cell And Gene Therapy Market Regional And Country Analysis

Companies Mentioned

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Global Cell and Gene Therapy Market Report 2020-2030: COVID-19 Impacts, Growth and Changes - GlobeNewswire

The hunt for effective therapies for solid tumors has heated up in early 2021 with a string of biotechs touting big investor checks and name-brand collaborations to chase those hard-to-treat lumps. Now, with one of its candidates already in the clinic for leukemia, Mana Therapeutics is taking a new round of funding to join the fray.

On Friday, Mana unveiled a $35 million Series A financing round that will help push the Boston-area biotechs lead candidate through a Phase I trial and could help the company secure an IND for an off-the-shelf allogeneic molecule targeting transplant-ineligible AML and solid tumors within the year.

The biotechs leading molecule, dubbed MANA-312, is already engaged in the Phase I study of patients with acute myeloid leukemia, myelodysplastic syndrome after undergoing an allogenic hematopoietic stem cell transplantation. Manas goal is to use its technology to create an inventory of off-the-shelf allogeneic products that can treat the majority of patients with certain targeted cancer indications using whats called a human leukocyte antigen matching system.

Its a different take on a similar line of attack for solid tumors: using the bodys natural immune system to educate healthy cells already in the body to target antigens on the surface of the tumors cancer cells without damaging the otherwise healthy cells. To do this, Mana uses an in-house platform called EDIFY, which it says leverages natural immune system pathways to educate T cells to target multiple cell surfaces and intracellular tumor-associated antigens.

Through multiple antigen targeting processes, the companys technology is designed to prevent the tumors immune escape, and it says the allogeneic method which uses healthy donor cells to create a master cell bank and is then used for specific therapies of attacking the solid cancer tumors could provide superior efficacy to single antigen and other cell therapy approaches.

MANA-312 also isnt the biotechs only candidate in the works. MANA-412 is a preclinical off-the-shelf allogeneic cell therapy for treating transplant-ineligible acute myeloid leukemia and solid tumors and could be ready for an IND filing by the end of the year, Mana said. The Series A round will help fund preclinical development for that candidate as well.

Mana was founded based on research and human proof-of-concept clinical trials conducted by Catherine Bollard of Childrens National Hospital and her colleagues at Johns Hopkins Medical Center. Those trials, in both solid and hematologic tumors, supported a strong safety profile, showed immunological anti-tumor activity and validated MANAs initial set of tumor antigens, the company said. Then Bollard co-founded the company with industry vet Marc Cohen. Ex-Gilead exec Martin Silverstein is the CEO.

The human proof-of-concept trials conducted by my team and colleagues showed potential for a nonengineered approach to educating T-cells to attack multiple tumor antigens, which MANA is expanding even further through refinement of the manufacturing process for an allogeneic product and application to a broader set of antigens in a variety of clinical indications and settings, Bollard said in a statement.

MANAs $35 million financing round was led by Cobro Ventures and Lightchain Capital and joined by LifeSci Venture Partners with other undisclosed investors.

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Mana joins the hectic fight against solid tumors with an 'off-the-shelf' candidate angling for an IND this year - Endpoints News

Dive Brief:

As the name suggests, biomolecular condensates are tiny concentrations of molecules found inside cells. Scientists have observed these clusters, which take the form of liquid-like droplets, for decades, but only recently determined that they help regulate cellular reactions and activities.

Like other cell-managing structures, researchers suspect that biomolecular condensates can give rise to a variety of diseases if they malfunction. This thinking has led to the formation of several new drug companies in the last couple years, with Dewpoint being the first to come out of stealth mode.

Dewpoint arrived in early 2019, backed by the venture capital firm Polaris Partners and a string of other investors. Company leadership said the initial focus would be cancer and neurodegenerative diseases, though other areas like immunology and infectious disease also appear to be on Dewpoint's radar. The Boston-based biotech quickly drew interest from pharmaceutical giants, too, with Bayer and Merck & Co. inking separate deals potentially worth $100 million or more.

With the Pfizer deal, Dewpoint joins a handful of companies targeting DM1. Audentes Therapeutics, a gene therapy developer now owned by Astellas, has been exploring two approaches to treat the disease. Vertex and CRISPR Therapeutics also recently expanded a gene editing partnership to include DM1 and Duchenne muscular dystrophy.

Faze Medicines has its sights set on DM1 as well. The Cambridge, Massachusetts-based biotech debuted in December with $81 million in Series A funding, which was supplied, in part, by some big pharmaceutical firms, including the Novartis Venture Fund and AbbVie Ventures.

Faze is also trying to find and develop treatments for ALS. Interim CEO Cary Pfeffer recently told BioPharma Dive that Faze may take aim at other neurological and neurodegenerative disorders, as well as cancer, immune diseases and viral illnesses.

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Dewpoint forges another big pharma partnership and a potential rivalry - BioPharma Dive

A little less than two years after bagging an extended $50 million Series C, IsoPlexis and its proteomic barcodes for personalized cancer care are back setting hooks to bring even more investors on board. And this time, theyve caught a big fish.

Perceptive Advisors is leading a $135 million Series D round for the company, the firms announced Thursday, comprising $85 million in equity and a $50 million line of credit. IsoPlexis plans to use the proceeds to expand commercial and R&D staff, increase operational capacity and accelerate product development.

The Branford, CT-based company works with cancer centers and biopharma companies in the US, Europe and China, using its biomarker-driven system designed to predict responses to such treatments and personalize treatment for patients. IsoPlexis employs a proprietary single-cell analysis tool to refine its immunotherapies.

We believe the future of advanced medicines will rely upon deeper access to in vivo biology for the development of new therapies and are excited to back the team at IsoPlexis, Perceptive portfolio manager Sam Chawla said in a statement.

Researchers have developed what they call proteomic barcode chips, which allow them to look at the entire complement of proteins within a patients cells. Its a process they say provides the mapping of new and accessible layers of biological data for every single cell, ultimately allowing for a better understanding of how individuals may respond to therapies.

Essentially, patients receive samples of these chips, which CEO Sean Mackay says is barcoded with antibodies. After the company receives the sample back, they place it into their system to see just how a persons immune system would respond to different treatments.

We call that the single-cell immune landscaping, Mackay told Endpoints News. What were able to do with that is find subsets of powerful immune cells that you typically miss in bulk profiling, sort of status quo, and that is a product that works on our instrument, basically a software-enabled system that reads out what the chips look like and what the proteins are per cell.

That software then lets IsoPlexis compare whats typically missed in that bulk profiling to long-term responder patients in several different fields like cancer immunotherapy, cell and gene therapy, Covid-19 and autoimmune disease, among other areas. IsoPlexis can then pick and choose the appropriate preclinical treatments and biomarkers in the clinic, packaging that info to pharma companies and academic labs.

Perceptive, historically a passive investor that enjoys clinical-stage investments and crossover rounds, has been fairly busy over the last year or so. It made its first foray into the company formation and Series A spaces in late 2019, setting up a $210 million early-stage VC fund with Xontogeny. Then last August, they launched their first in-house start-up in China, followed by a $310 million raise a few months later. Perceptives third SPAC also filed for an IPO in late July.

Other new investors included Ally Bridge Group and funds and accounts managed by BlackRock. Unnamed existing investors also participated in the round.

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IsoPlexis scores big backer for personalized protein 'barcodes' as Perceptive jumps on board new funding round - Endpoints News

LONDON--(BUSINESS WIRE)--The soft tissue repair market is poised to grow by USD 10.44 billion during 2020-2024, progressing at a CAGR of over 11% during the forecast period.

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The report on the soft tissue repair market provides a holistic update, market size and forecast, trends, growth drivers, and challenges, as well as vendor analysis.

The report offers an up-to-date analysis regarding the current global market scenario and the overall market environment. The market is driven by the rising incidence of accidental injuries.

The soft tissue repair market analysis includes Product segment and Geography Landscape. This study identifies the growing demand for gene therapy as one of the prime reasons driving the soft tissue repair market growth during the next few years.

This report presents a detailed picture of the market by the way of study, synthesis, and summation of data from multiple sources by an analysis of key parameters.

The soft tissue repair market covers the following areas:

Soft Tissue Repair Market SizingSoft Tissue Repair Market ForecastSoft Tissue Repair Market Analysis

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Global Soft Tissue Repair Market- Featuring 3M Co., Arthrex Inc., and Baxter International Inc. Among Others - Business Wire

In an effort to crack the code of hard-to-treat solid tumors, biopharma has tried numerous pathways to effectively target those masses without damaging healthy tissues. Phillys Carisma Therapeutics thinks it has a winner with its macrophage cell-based CAR-M candidates, and now its taking a new flush of investor cash to try one in the clinic.

Carisma has scored a $47 million Series B round to take its lead candidate, anti-HER2 CAR-M tumor fighter CT-0508, into a Phase I trial as well as advancing the rest of its preclinical macrophage pipeline. Carisma has keyed in on the use of targeted macrophage cells and vectors to penetrate the environment of solid cancer tumors without hurting health tissues a puzzle in the solid tumor field.

CT-0508s early-stage study will turn out the first human data for a CAR-M therapy based on those macrophage cells, Carisma said. CEO Steven Kelly told Endpoints News his company could offer an effective and safer way to target tumors and warm them up for the immune system to attack.

There are a number of characteristics about macrophages that would lend themselves towards applications of solid tumors and other indications, but what were focused on is solid tumors, Kelly said. Macrophages are preferentially recruited into a tumor microenvironment, and lymphocytes like the CAR-T approaches are actively excluded. So we think that we have an advantage by overcoming trafficking limitations to solid tumors.

This has been the sticking point for the industry: therapeutics that can invade the walls which surround cancerous tumors without damaging otherwise healthy cells.

Kelly is confident that Carismas technology will ultimately decipher how to do just that.

Once it starts eating (the cancerous tumor cells), the macrophages will start producing cytokines that effectively warm up that environment and convert an immunologically cold tumor into a warm or hot tumor and recruit in other cells, like T cells for example. So that last element is really unique to macrophages due to the antigen presentation capability, he said. They engage in cells directly, they warm up the tumor microenvironment, and they generate a true adaptive immune response. Thats how we think of ourselves and how were differentiated in the cell therapy space.

Kelly said it was a bit premature to know when Carisma would begin public readouts of the data surrounding its macrophage therapeutics, but he hoped they would be able to do so by the middle of this year.

The total capital Carisma has raised since its Series A financing in 2018 now totals roughly $109 million, and is a key step in moving the company from effectively a discovery-stage company to a clinical-stage company, Kelly said.

A lot of effort has gone into building this company. We had to transition from a bench project at (the University of) Penn, we had to demonstrate all the things necessary to get an IND declared (so) safety and efficacy we had to develop a GMP manufacturing process, Kelly said. All those were effectively developed to FDA satisfaction, and were moving into the clinic now.

Investors in the Series B financing are led by Symbiosis II, with subsequent investment from Solasta Ventures and Livzon Pharmaceutical Group. Additionally, Carisma said, existing investors AbbVie Ventures, HealthCap, Wellington Partners, IP Group, TPG Biotech, Agent Capital, and MRL Ventures Fund contributed to the new round of funding.

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Looking to solve the solid tumor puzzle box, Carisma aims to take 'CAR-M' groundbreaker into early-stage trial - Endpoints News

Investors like to see big plans, and Kevin Judice has plenty. The DiCE Molecules CEO is plotting a clinical trial launch for the biotechs lead small molecule for psoriasis and wants to double the staff in the next year and a half.

On Friday, those big plans landed him an $80 million Series C round.

Were very excited about it, he said of the raise led by RA Capital Management.

The round comes around two years after a $50 million Series B. While the B round was used for optimizing technology and building a pipeline, Judice says the Series C will propel the biotechs IL-17 antagonist to the clinic and fund the development of two other undisclosed programs.

This new capital allows us to expand our reach and get at more targets and have more opportunities to make high impact, Judice said.

DiCEs development process revolves around its DNA-encoded library. Such libraries allow researchers to screen millions even billions of compounds in parallel, using DNA tags that Judice compared to barcodes, which tell you what the constituent pieces of a molecule are.

Usually you do some kind of screen, like a high-throughput screen, or a DNA-encoded library screen, something like that, and you get a few hits. And then theres a long phase of just lab chemistry, where youre making individual compounds and trying to progress those hits, those initial binders, to something thats closer to a drug, Judice said.

That hit-to-lead phase is typically labor-intensive and slow, the CEO said. But DiCEs approach accelerates that work by using a smaller DNA-encoded library much smaller but richer in information, Judice said to screen in different ways after getting a hit.

What were actually looking for is the difference between just binding and something that is functional, Judice said.

DiCEs lead program is an agonist for cytokine receptor IL-17, which is implicated in diseases like psoriasis and psoriatic arthritis. Current antibody treatments targeting IL-17 are quite effective at treating psoriasis, but they are injectable and lack in convenience. DiCEs candidate would be oral, and the biotech is hoping to top the efficacy of Amgens already approved oral PDE4 inhibitor Otezla.

What were working on is an oral that will work as well as the anti-IL-17 antibodies. So it combines the convenience and safety of something like Otezla with the efficacy of an antibody like Cosentyx, Judice said. The antibodies tell us that IL-17 is exactly the right target.

Since 2017, DiCE has grown from a seven-person, peanut-sized company to a 29-person staff. And in the next 18 months, Judice is looking to bring that number to 58. The biotech inked a $2.3 billion discovery pact with Sanofi years ago, and is currently partnering with them on an I-O small-molecule program that Judice says isnt far behind the IL-17 candidate.

We should be ready to go public with more data on earlier programs over the course of the next 12 months. And then Im really excited about the opportunity to grow the pipeline by adding new programs to it, he said. Thats one of the things that is particularly great, from my perspective, about having RA Capital lead this round.

In addition to RA, Eventide Asset Management, New Leaf Venture Partners, Soleus Capital, Driehaus Capital Management, Osage University Partners and Asymmetry Capital Management, Northpond Ventures, Sands Capital, Sanofi Ventures, Alexandria Venture Investments, Altitude Ventures and Agent Capital also chipped into the Series C.

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DiCE gets its 'library' card ready as it speeds development of DNA database-derived molecules with more investor cash - Endpoints News

Dec. 28 (UPI) -- Fewer than 2% of people with treatment-resistant high blood pressure are checked for a hormone disorder that can drive blood pressure higher, a study published Monday by the Annals of Internal Medicine found.

In people with primary aldosteronism, the adrenal gland produces too much of a hormone called aldosterone, causing increases in blood pressure, often to unhealthy levels, according to the Mayo Clinic.

Professional guidelines recommend checking those with high blood pressure that doesn't respond to treatment for the hormonal disorder, but researchers found the tests often are not performed.

"While primary aldosteronism is a common cause of difficult to control hypertension, it is under-diagnosed," study co-author Dr. Jordana Cohen told UPI.

"Patients who were tested in our study were more likely to be treated with the appropriate medications ... and to have better blood pressure control over time, said Cohen, an assistant professor of medicine and epidemiology at the University of Pennsylvania.

Many people who don't respond well to commonly used blood pressure control medications, including beta blockers and ACE inhibitors, are found to have the hormone disorder, Cohen and her colleagues said.

The condition has been linked with a four- to 12-fold increased risk for cardiovascular events, such as heart attack and stroke, compared to those with high blood pressure due to other causes, the researchers said.

However, the disorder can be effectively treated with drugs called mineralocorticoid receptor antagonists, or MRAs, including spironolactone and eplerenone, or surgery, according to Cohen.

For this study, she and researchers at the University of Pennsylvania, Stanford University and the University of Michigan reviewed data from the Veterans Health Administration for more than 269,000 veterans with apparent treatment-resistant high blood pressure.

Treatment-resistant hypertension was defined as either two blood pressures of at least 140 systolic or 90 diastolic at least one month apart. Patients also had to be receiving treatment with at least three blood pressure drugs, including a diuretic, or with at least four different types of blood pressure drugs, the researchers said.

Just under 2% of patients with treatment-resistant high blood pressure underwent guideline-recommended testing for the hormone disorder, the data showed.

Testing rates ranged from 0% to 6% at centers included in the study and did not correlate with the number of patients with treatment-resistant hypertension, the researchers said.

And just 15% of the patients were on an MRA drug, the data showed.

Patients tested for the hormone condition were more likely to receive treatment with MRAs and have better long-term blood pressure control, according to the researchers.

Testing rates also did not change meaningfully over nearly two decades of follow-up despite an increasing number of guidelines recommending testing for the disorder in this population, they said.

"If you are on three or more medications for management of your blood pressure, ask your doctor if they think you might benefit from testing or from being treated with an MRA," Cohen said.

"Not all patients are appropriate to be tested or treated with MRAs, but most people with treatment resistant hypertension are," she said.

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Study: Too few with high blood pressure tested for hormone disorder - UPI News

Over the last several years, parents from across the country have appeared on television and news outlets to raise money to develop treatments for their children with rare genetic disorders.

Many of these families, including one from Kansas City, Missouri, have pinned their hopes on one Texas researcher, Dr. Steven Gray of the University of Texas Southwestern Medical Center in Dallas, who claims to be on the verge of treating a number of rare conditions. They've raised millions of dollars to fund his research, although breakthroughs haven't happened to the extent many had hoped.

While gene therapy holds great promise, the growing trend of family-funded research concerns some medical ethicists, who say that suggestions to parents that treatments may be imminent can raise thorny ethical issues.

These are parents. They are desperate. says Mayo Clinic bioethicist Megan Allyse. They are willing to try almost anything. They are in a pretty vulnerable position for somebody to come along and say, Give me your money, and I can make this better.

Further complicating the picture is the recent entry into the field of a private company, Taysha Gene Therapies, which says it will accelerate Grays research. That has divided his supporters and raised concerns about families who might be left behind.

Kim Fry, of Kansas City, Missouri, has a video on her phone of her son, Charlie, that was made in 2018 when he was 5 months old. It shows her bright-eyed boy gently shaking in his father's lap, as if shivering from cold.

Those little tremors sent the family on a year-long odyssey that led to a frightening diagnosis. A genetic test showed Charlie had an incredibly rare mutation in a single gene, SLC6A1. The mutation typically causes intellectual disabilities and epilepsy starting around the age of three-and-a-half that can severely affect patients for the rest of their lives.

Doctors told Kim and Nate there was no treatment available to help their son.

At that moment, you just feel crushed and kind of begin grieving for the life you think your childs going to have, Kim said.

Rare genetic disorders have generally received little attention from biotech companies because the markets for treatments are so small.

But shortly after the diagnosis, Kim met Amber Freed, a mother from Denver who seemed to have found a solution. After her son, Maxwell, was diagnosed with the same mutation a year earlier, Freed met Gray, a molecular biologist who had focused on gene therapy while doing a post-doctoral fellowship at UNC Chapel Hill.

Gray was developing treatments or even possible cures for conditions caused by single-gene mutations, and he had agreed to work on SLC6A1. But it would be up to Freed to provide Gray with the millions of dollars he would need to do this work.

With the possibility of a treatment suddenly on the table, Kim and Nate immediately joined Amber in raising money through the organization she started, SLC6A1 Connect, and their own campaign, A Cure For Charlie. Their goal was to create a treatment and bring it to clinical trial before Charlie turned three-and-half, hoping to block the severe effects of the mutation and giving him a chance to live a regular life.

Once the funding is there, then all the science is going to move into place, so really the only hurdle that were facing right now between us and the cure is the funding, Nate said in fall of 2019.

Family fundraisers

Family fundraisers are a departure from how medical research is usually funded, typically through the National Institutes of Health, large foundations and advocacy groups.

Nevertheless, families like the Frys have appeared on news outlets and in publications throughout the country in recent years, from Good Morning America and ABC News to People magazine and countless local news shows, to raise money for research.

The children have been diagnosed with many different genetic disorders, but their stories are similar. They all have rare gene mutations that will lead to serious mental or physical declines or early death, and their parents are pinning their hopes on Gray to develop treatments.

They call themselves the Steve Gray Parents.

Gray himself has appeared in articles and videos, including one produced by UT Southwestern about Willow Canaan, a girl from Mississippi who has multiple sulfatase deficiency.

A lot of the families that we interact with, they are coming to us with really sick kids. I think knowing their story, knowing that one child, gives us a face, gives us a mission that if we can move fast enough theres hope that we could treat and we make things different for that specific child, Gray said in one video.

Gray became a go-to researcher for rare disease families after his treatment of Hannah Sames, a girl from New York with a degenerative genetic disorder called giant axonal neuropathy.

In a 2016 clinical trial, Sames was injected with a manufactured virus that contained a working copy of the gene that was mutated. Through this adeno-associated virus delivery method, the normal gene would take over from the mutated one and stop the degeneration from happening.

The treatment slowed the progress of Hannahs disease, according to Gray, but it wasnt a cure. In a 2019 interview on Connecticut Public Radio, Hannahs mother, Lori, said the family was seeking additional treatment.

But Gray thinks the same method could be used to treat and possibly cure all kinds of genetic disorders, including the SLC6A1 mutation

Were due for another leap in technology, Gray told KCUR in 2019. Were going to have a better virus technology, better ways to deliver genes, and I can see that just making a further leap for the whole field.

Gray, who says he has been involved in developing treatments for two dozen diseases, has accepted money from families to pay for the high costs of manufacturing viruses, doing toxicology studies and running clinical trials. Many of these family groups had raised more than a $1 million each from their friends, relatives and neighbors.

Though these families have been effective at raising money, bioethicist Allyse worries that without the peer review process that traditional funders use, they may not be in the best position to decide what research is likely to get results.

The potential problem with going around that process is that its possible to sort of go down avenues that are less supported by the literature, that are less in line with the scientific consensus, Allyse said.

But those doubts have done little to discourage dedicated parents like Amber Freed.

In early December 2019, Freed hosted the second annual SLC6A1 research symposium in a hotel conference room in downtown Baltimore. Freed quit her job in finance after her sons diagnosis to dedicate herself to advancing a treatment, and she began organizing annual SLC6A1 research symposiums in 2018 to drum up interest in the work.

Shes also held charity golf tournaments, set up fundraising campaigns with companies like Amazon and Pizza Hut, and helped arrange the creation of genetically altered mice in China for research.

During the last two years, Freed has cultivated relationships with genetic researchers from all over the world, and as the sleepy scientists who traveled to Baltimore to take part in the symposium wandered into the conference room early on a Friday morning, she greeted them like family, with big hugs and smiles.

Alex Smith

Despite Freeds seemingly endless enthusiasm, she made clear in her welcoming speech to the scientists that, unlike them, her involvement in SLC6A1 research didnt happen by choice.

But to be honestI dont want to be here, read a slide projected behind her at the end of her remarks.

Some improvements but no 'home run'

Toward the end of the day, Gray took the podium to deliver an update on research from his lab. For many in the audience, this was the days main event.

His teams early research using the treatment showed some improvements in motor and behavioral skills in young, genetically altered mice that were treated before symptoms had appeared. But there was no change in mice that already had symptoms.

I think treating at an early age, were seeing some signs of improvements and some nice signals that our vector is doing something positive, but, you know, its not a home run, Gray explained.

Though it wasnt the result Freed dreamed of, she was encouraged that the research appeared to be on the right path.

Gray insisted he had tried to be careful about managing expectations for families funding his work, but between symposium talks, he also said he had recently shifted course on working with them.

Im really having to say no a lot now, Gray told KCUR. Im kind of moving into a point where we were trying not to say no, and we were trying to work on everything that the science made sense. But there is a point where you just have to say, You know Ive got to focus on what Im doing, and theres a limit.

While Grays work did lead to a treatment for Hannah Sames, similar breakthroughs havent come in time for other families.

Laura King Edwards of Charlotte, North Carolina, started working with her family to raise money for Grays work after her baby sister, Taylor, was diagnosed with a form of a rare disorder called infantile Batten disease in 2006.

The family didnt have a lot of hope the research would lead to a treatment in time to help Taylor, and she died two years ago at age 20.

Edwards says that looking back, she sometimes regrets all the time she spent running a fundraising organization.

Id spend hours up at night dealing with tech issues on our website, for example, or responding to emails from people all over the world, knowing that thats time that maybe I couldve spent with my little sister while she was still here, Edwards says.

Nevertheless, even after Taylor was gone, her family continued to support Grays work through their organization, Taylor's Tale.

A new player

Not long after the conference in Baltimore, however, the race for a SLC6A1 treatment slowed to a crawl.

When the COVID-19 pandemic hit, scientific studies and medical trials across the country were stopped and research funds were directed to coronavirus research.

The therapists who work with Amber and Kims sons were unable to meet with them in person, and the boys started to backslide on some of their developmental progress.

Then, after the initial waves of the coronavirus subsided, hopes for Grays research came roaring back to life when a new company, Taysha Gene Therapies, announced it would partner with UT Southwestern, offering a boost to the research and development beyond what families could provide.

They could get it to a certain place, said Taysha founder R.A. Session II, But when it needs to get to kind of the meaningful level in order to get it into late-stage clinical trials, this is where they just dont necessarily have the capability. And so I think this is where you would see programs then transitioning into a companys hands in order to kind of pursue them and move them forward.

In April, Taysha announced a partnership with UT Southwestern that would fund Grays research and work to move it more quickly into clinical trials and possible treatment. Gray was named chief scientific officer.

The company said the family fundraising would no longer be needed.

For some parents, like Doug and Kasey Woleben of Dallas, that was great news. Theyve raised around $1 million for research to treat Leigh syndrome, which affects their 8-year-old son, Will.

We were excited, thrilled to know that were now off the hook for millions and millions and millions of dollars. And that Taysha and UT Southwestern are trying to push this program and move it forward as quickly as possible. So for us, it was a miracle, Kasey said.

But Taysha's involvement and its timeline have brought disappointment for other families. The company's first clinical trials, to treat a mutation that causes Tay Sachs disease, were planned to start in Canada at the end of 2020 but only received approval from the Canadian government this week.

The company says it plans to seek permission to test treatments for three other conditions, including the SLC6A1 mutation, by the end of 2021, but it has not announced any dates for beginning trials.

For Amber Freed and Kim Fry, Tayshas timeline is problematic. Both of their sons were expected to exhibit epilepsy symptoms before the end of next year and so they would see little benefit from treatment initiated after that.

Im very disappointed, Freed said in September. If you had asked me this time last year, I would have fully expected to be in a clinical trial right now.

Different priorities

Session insists that Tayshas timeline and priorities on are based on what the research shows is safe and effective.

Weve allowed the science to kind of move forward at the pace the science moves, Session says. Then we move it forward into the clinic based on that science.

But to Freed, the goal of fast-tracking to trial, even one that would have only resulted in a slight improvement for her son, appears much less likely now that Taysha is involved.

Once you hand over the reins to a biotech, you lose decision-making power as a nonprofit organization, and you abide by their timeline and not necessarily your own, Freed said. In my case, we are racing to get this therapy into children like Maxwell and Charlie as quickly as possible, so we need it done tomorrow.

For other Gray supporters, however, the future is even less clear. Tayshas development pipeline does not include treatments for some of the conditions that Gray had previously been working with families to develop, including Charcot-Marie Tooth, Krabbe disease and multiple sulfatase deficiency.

UT Southwestern researchers will continue to research those conditions, according to a university spokesman, and Taysha says it plans to expand its pipeline in the future.

Terry Pirovolakis, who had enlisted Grays help to develop a treatment for spastic paraplegia 50, which affects his son, Michael, will not be involved with Tayshas work. Hell only continue to work with UT Southwestern directly.

From my perspective, it was, thats great. Tayshas gonna come in and maybe save the world, but I dont want to be part of it 'cause theyve got a lot of stuff they gotta work out, and Im not going to wait around for them to figure it out, Pirovolakis said.

Pirovolakis, who lives in Toronto, has raised more than $1.5 million since May 2019 through his online campaign, Cure Michael, which was the most successful GoFundMe campaign in Canada last year.

Expectations vs. reality

He says that while he has been comfortable working with Gray, he believes that drug companies, which depend on the involvement of families for rare disease research, can mislead parents about what might be possible for their children.

The industry, as a whole, I think, maybe sets expectations that are higher than reality," Pirovolakis said. "We see these presentations at the conferences of these kids doing amazing things, like a 4-year-old that has no brain function pretty much, going to school two years later. Its remarkable.

"But that was five or 10 years of research. So I think that expectations from the industry are maybe what cloud us as parents in the hope that something amazing is gonna happen for our kids.

CureCMT4J, a foundation created to advance research on Charcot-Marie Tooth by parent advocate Jocelyn Duff, an early supporter of Gray, also is no longer involved with the researchers work. Duff said the organization had moved in a different direction, but she declined the provide details. The group had raised $1.3 million as of fall 2020.

Some ethicists have also raised questions about the costs of rare disease treatment, and they point to a drug previously developed by members of the Taysha team as a prime example of their concern.

Several members of the Taysha team, including Session, directors Sean Nolan and Phillip Donenberg, and others, earned their reputation for success as part of a AveXis, a company that developed the breakthrough treatment for spinal muscular atrophy, Zolgensma.

Zolgensma was introduced by drug giant Novartis last year with a price tag of more than $2.1 million, making it the most expensive drug in the country.

On the one hand, you could say thats a winning team, said Megan Allyse. On the other hand, you could say is that the team you want to be on if what youre trying to do is generate not just effective treatments, but accessible treatments?

Session says that Taysha currently has no plans regarding the pricing or accessibility of any treatments the company might develop.

We should be so blessed to be able to have a discussion on pricing because then were talking about an approved therapy, Session says. But were not there yet. So what I would say is the company is focused on getting these drugs into patients effectively and safely as efficiently as possible.

Taysha announced in November that it raised more than $275 million in private financing and an initial public offering.

For Kim Fry and Amber Freed, however, the focus is still very much on what can be done for their sons.

The women are continuing to raise money, but they have shifted to other researchers and technologies. And they have adjusted their expectations.

Frys son, Charlie, started having more significant seizures earlier in the year and is now taking medication to reduce them.

She still thinks a treatment within the next year or two could help her son and others like him, although not in the way she had once imagined.

It may not be a 100% home run where they live 100% the life we hoped. But theyll still have a better life than they are living today, Fry said. I lose sleep every night over the thought that it might be too late, but Im still hopeful that there will be benefit for them.

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These Families Raised Millions To Fund Treatment For Their Kids' Genetic Disorders. It Hasn't Happened. - KCUR

TSHA-101 will be the first bicistronic vector to enter a first-in-human clinical study, which is a significant milestone for Taysha and for the field of gene therapy, said Suyash Prasad, MBBS, M.SC., MRCP, MRCPCH, FFPM, Chief Medical Officer and Head of Research and Development of Taysha. GM2 is a devastating lysosomal storage disease with no approved treatments and todays CTA approval marks a formative moment for children suffering from this rapidly progressive and fatal disease.

The trial will be a single arm, open-label Phase 1/2 trial evaluating the use of TSHA-101 for the treatment of infants with GM2. The study will be sponsored by Queens University and led by Jagdeep S. Walia, MBBS, FRCPC, FCCMG, Clinical Geneticist and Associate Professor Head, Division of Medical Genetics (Department of Pediatrics) at Queens, and Director of Research (Department of Pediatrics), at the Kingston Health Sciences Centre.

Preclinical evidence to date supports our belief that TSHA-101, when given intrathecally as a bicistronic transgene packaged into a single AAV9 vector, has the potential to address the lysosomal enzyme deficiency, to change the disease trajectory and to improve patient survival, said Dr. Jagdeep S. Walia. We are pleased to have the support of Health Canada as we continue to advance TSHA-101.

Todays CTA approval is a culmination of our teams and Dr. Walias tireless efforts and a momentous occasion for children affected by GM2 along with their parents and caregivers, said RA Session II, Founder, President and CEO of Taysha. We are grateful to our partners at Queens University for their work to advance this gene therapy into the clinic.

About GM2 Gangliosidosis

GM2 gangliosidosis is a rare and fatal monogenic lysosomal storage disorder and a family of neurodegenerative genetic diseases that includes Tay-Sachs and Sandhoff diseases. The disease is caused by defects in the HEXA or HEXB genes that encode the two subunits of the -hexosaminidase A enzyme. These genetic defects result in progressive dysfunction of the central nervous system. There are no approved therapies for the treatment of the disease, and current treatment is limited to supportive care.

About TSHA-101

TSHA-101 is an investigational gene therapy administered intrathecally for the treatment of infantile GM2 gangliosidosis. The gene therapy is designed to deliver two genes HEXA and HEXB driven by a single promoter within the same AAV9 construct, also known as a bicistronic vector. This approach allows the simultaneous expression of a 1:1 ratio of the two subunits of protein required to generate a functional enzyme. It is the first and only bicistronic vector currently in clinical development and has been granted Orphan Drug and Rare Pediatric Disease designations by the U.S. Food and Drug Administration (FDA).

About Taysha Gene Therapies

Taysha Gene Therapies (Nasdaq: TSHA) is on a mission to eradicate monogenic CNS disease. With a singular focus on developing curative medicines, we aim to rapidly translate our treatments from bench to bedside. We have combined our teams proven experience in gene therapy drug development and commercialization with the world-class UT Southwestern Gene Therapy Program to build an extensive, AAV gene therapy pipeline focused on both rare and large-market indications. Together, we leverage our fully integrated platforman engine for potential new cureswith a goal of dramatically improving patients lives. More information is available at http://www.tayshagtx.com.

Forward-Looking Statements

This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as anticipates, believes, expects, intends, projects, and future or similar expressions are intended to identify forward-looking statements. Forward-looking statements include statements concerning or implying the potential of our product candidates, including TSHA-101, to positively impact quality of life and alter the course of disease in the patients we seek to treat, our research, development and regulatory plans for our product candidates, the potential for these product candidates to receive regulatory approval from the FDA or equivalent foreign regulatory agencies, and whether, if approved, these product candidates will be successfully distributed and marketed. Forward-looking statements are based on managements current expectations and are subject to various risks and uncertainties that could cause actual results to differ materially and adversely from those expressed or implied by such forward-looking statements. Accordingly, these forward-looking statements do not constitute guarantees of future performance, and you are cautioned not to place undue reliance on these forward-looking statements. Risks regarding our business are described in detail in our Securities and Exchange Commission (SEC) filings, including in our Quarterly Report on Form 10-Q for the quarter ended September 30, 2020, which is available on the SECs website at http://www.sec.gov. Additional information will be made available in other filings that we make from time to time with the SEC. Such risks may be amplified by the impacts of the COVID-19 pandemic. These forward-looking statements speak only as of the date hereof, and we disclaim any obligation to update these statements except as may be required by law.

View source version on businesswire.com: https://www.businesswire.com/news/home/20201221005197/en/

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Taysha Gene Therapies Announces Queen's University's Receipt of Clinical Trial Application Approval from Health Canada for Phase 1/2 Clinical Trial of...

The argument for continued investment

C> is a high potential and maturing sector, and is an already crowded environment, playing host to numerous start-ups and now, through M&A, recognised big pharma firms. Much like the rush to find a COVID-19 vaccine that dominates headlines worldwide, not every company involved will be able to succeed.

But finnCaps finnLife watch list of 50 leading AIM-listed biotech companies demonstrates that there is room for numerous companies to contribute to, and profit from, C>. Examining three entirely different approaches to CAR-T therapy, it is possible to see just how much space there is for this exciting sector, therefore displaying the case for continued investment.

Innovative CAR-T therapy demonstrates the depth of C> potential

CAR-T therapy in its existing form is a relatively new and specialised approach at treating cancer. It takes T cells from a patients bloodstream and genetically modifies them in a laboratory. These T cells are then injected back into the bloodstream with the aim of targeting and killing cancer cells.

While it has been shown to be an effective treatment, there are risks and side effects. One is the two-step autologous process (the slow time it takes for cell expansion sometimes as long as two weeks) while another is cytokine release syndrome (CRS), which occurs when cytokine molecules are inadvertently released, but too quickly to target just the tumours and instead target healthy cells.

The next generation of CAR-T treatments shows that there is space for a multitude of start-ups to be active in the C> space as they all help find varied solutions to these problems without negating the effectiveness of CAR-T.

One example is Horizon Delivery, a company that is developing its CYAD-02 project, which will help transport T cells more effectively to the tumour via the use of SMARTvector products.

The product underwent its first phase 1 trial test in January 2020 with a patient who was suffering from acute myeloid leukaemia. Horizon Delivery is also an industry leader in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screenings, meaning they can identify key genes or genetic sequences that draw out specific functions of a cell type from thousands of potential variants.

In a cancer context, this means they can route out and exclusively eliminate problematic cells that may have shown signs theyd resist a future cancer treatment.

Another example is Maxcyte, a global cell- based therapies and life sciences company that is developing its CARMA process, where a patients peripheral blood mononuclear cells (PBMCs) are removed and modified. The modified cells can then be used to target an array of different cancers.

Currently the company is conducting a phase 1 trial for advanced ovarian cancer in a dose escalation trial that will treat four separate cohorts the fourth of which was administered in March 2020.

Another example which shows the versatility of new CAR-T innovation is provided by Oxford Biomedica, a gene and cell therapy company specialising in the development of gene-based medicines.

Rather than a contained project or platform, its contribution to CAR-T is through a contract manufacturing development organisation. Collaborating with pharma companies, Oxford Biomedica uses its infrastructure to produce other companies licensed products, including Novartis Kymriah treatment (alongside other undisclosed CAR-T-related products).

With fast-moving innovation finally allowing multiple C> treatments to gain regulatory approval, along with a huge pipeline of upcoming therapies and an influx of funding and M&A activity, investing in C> no longer entails taking a bet on potential the future is finally here.

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After years of potential, cell and gene therapy is ready for the pharmaceutical mainstream - PMLiVE

Dublin, Dec. 23, 2020 (GLOBE NEWSWIRE) -- The "Regenerative Medicine in Pharma - Thematic Research" report has been added to ResearchAndMarkets.com's offering.

Regenerative medicine is a multidisciplinary field that seeks to develop the science and tools that can help repair, augment, replace, or regenerate damaged or diseased human cells, tissues, genes, organs, or metabolic processes, to restore normal function. It may involve the transplantation of stem cells, progenitor cells, or tissue, stimulation of the body's own repair mechanisms, or the use of cells as delivery vehicles for therapeutic agents such as genes and cytokines.

It is widely anticipated that Gene therapy is the most valuable regenerative medicine sector however, this market is also expected to be slowed down by high cost of therapies, which may limit its accessibility.Existing programs will facilitate the approval and development of regenerative medicines, however, a reimbursement system especially for curative therapies is warranted.

The Regenerative Medicine in Pharma report combines primary research from a cross-specialty panel of experts with in-house analyst expertise to provide an assessment of the development landscape.

This report assesses -

Scope

Key Topics Covered:

For more information about this report visit https://www.researchandmarkets.com/r/7trz9r

About ResearchAndMarkets.comResearchAndMarkets.com is the world's leading source for international market research reports and market data. We provide you with the latest data on international and regional markets, key industries, the top companies, new products and the latest trends.

Research and Markets also offers Custom Research services providing focused, comprehensive and tailored research.

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Regenerative Medicine in Pharma 2020 - Opportunities, Challenges, and Unmet Needs - GlobeNewswire

Global Gene Therapy for Rare Disease Market Report, Sales and Consumption Status and Prospects Professional Research, the report classifies the Global Gene Therapy for Rare Disease Market in a precise manner to offer detailed insights about the aspects responsible for augmenting as well as restraining market growth.

Gene Therapy for Rare Disease Market report provides a thoroughly researched abstract of the key players with considerable shareholdings at a global level regarding demand, sales, and income by providing better products and services. Research Report outlines a forecast for the Gene Therapy for Rare Disease market between 2020 and 2027. In terms of value, the Gene Therapy for Rare Disease industry is expected to register a steady CAGR during the forecast period.

In recent past, most of the gene therapies received orphan drug designations. Orphan drugs are generally defined as those medicines with one or more indications approved under the Orphan Drug Act of 1983. The Orphan Drug Act supports the development of innovative treatments for rare disease patients. The creation of the orphan drug designation with the passage of the Orphan Drug Act in 1983 has facilitated the development and approval of drugs for rare diseases and 2017 and 2018, were marked by the highest number of orphan drug and indication approvals to date. Production of gene therapies is associated with use of high-end technologies, high research and development costs, and skilled scientists and researchers, which reflects in high prices of these therapies.

Note: *The Download PDF brochure only consists of Table of Content, Research Framework, and Research Methodology

Get PDF Brochure Of This Research Report @ https://www.coherentmarketinsights.com/insight/request-pdf/2321

The key players profiled in this report include: Kite Pharma, Inc. (Gilead Sciences, Inc.), Novartis International AG, Juno Therapeutics Inc. (Celgene Corporation), Bluebird Bio, Inc., Spark Therapeutics, Inc., uniQure N.V, Orchard Therapeutics Plc., PTC Therapeutics, Inc., and BioMarin Pharmaceutical Inc.

Regions included:

o North America (United States, Canada, and Mexico)

o Europe (Germany, France, UK, Russia, and Italy)

o Global (China, Japan, Korea, India, and Southeast Asia)

o South America (Brazil, Argentina, Colombia)

o The Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria, and South Africa)

Key Benefits:

o This study gives a detailed analysis of drivers and factors limiting the market expansion of Gene Therapy for Rare Disease

o The micro-level analysis is conducted based on its product types, end-user applications, and geographic

o Porters five forces model gives an in-depth analysis of buyers and suppliers, threats of new entrants & substitutes and competition amongst the key market players

o By understanding the value chain analysis, the stakeholders can get a clear and detailed picture of this Gene Therapy for Rare Disease market

Table of Contents

Report Overview: It includes the Gene Therapy for Rare Disease market study scope, players covered, key market segments, market analysis by application, market analysis by type, and other chapters that give an overview of the research study.

Executive Summary: This section of the report gives information about Gene Therapy for Rare Disease market trends and shares, market size analysis by region and analysis of Global market size. Under market size analysis by region, analysis of market share and growth rate by region is provided.

Profiles of International Players: Here, key players of the Gene Therapy for Rare Disease market are studied on the basis of gross margin, price, revenue, corporate sales, and production. This section gives a business overview of the players and shares their important company details.

Regional Study: All of the regions and countries analyzed in the Gene Therapy for Rare Disease market report is studied on the basis of market size by application, the market size by product, key players, and market forecast.

The research study can answer the following Key questions:

What will be the progress rate of the Gene Therapy for Rare Disease Market for the conjecture period, 2020-2027?What are the prominent factors driving the Gene Therapy for Rare Disease Market across different regions?Who are the major vendors dominating the Gene Therapy for Rare Disease industry and what are their winning strategies?What will be the market scope for the estimated period?What are the major trends shaping the expansion of the industry in the coming years?What are the challenges faced by the Gene Therapy for Rare Disease Market?

Major Highlights of TOC:

Chapter One: Global Gene Therapy for Rare Disease Market Industry Overview

1.1Gene Therapy for Rare Disease Industry

1.1.1 Overview

1.1.2 Products of Major Companies

1.2Gene Therapy for Rare Disease Market Segment

1.2.1 Industry Chain

1.2.2 Consumer Distribution

1.3 Price & Cost Overview

Chapter Two: Global Gene Therapy for Rare Disease Market Demand

2.1 Segment Overview

2.1.1 APPLICATION 1

2.1.2 APPLICATION 2

2.1.3 Other

2.2 Global Gene Therapy for Rare Disease Market Size by Demand

2.3 Global Gene Therapy for Rare Disease Market Forecast by Demand

Chapter Three: Global Gene Therapy for Rare Disease Market by Type

3.1 By Type

3.1.1 TYPE 1

3.1.2 TYPE 2

3.2Gene Therapy for Rare Disease Market Size by Type

3.3Gene Therapy for Rare Disease Market Forecast by Type

Chapter Four: Major Region of Gene Therapy for Rare Disease Market

4.1 Global Gene Therapy for Rare Disease Sales

4.2 Global Gene Therapy for Rare Disease Revenue & market share

Chapter Five: Major Companies List

Chapter Six: Conclusion

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Gene Therapy for Rare Disease Market Prospects Pinpoint Higher Traction from Developed Nations during 2020-2026 | Coherent Market Insights | Kite...

Global Gene Therapy Market Research Report provides an complete recent proceeding in the market. The analysis supplies important data with figurative tables, graphs, charts, and statistics, an in-depth analysis of the market. The analysis highlights this markets fundamental dynamics for the forecast period (2020-2026), involving the trends, opportunities, restraints, and a lot more. The Gene Therapy analysis introduces a thorough evaluation to forecast the current market size, share, value, volume, gross sales, drivers, restraints, opinions by industry experts, and invaluable insights by the industrys prospective rise.

Every section of this report particularly consists of the research key elements of the market. The Gene Therapy industry dynamics segment permeates deep by the drivers, restraints, trends, and opportunities from this market. The quantitative and qualitative analysis, we help you with detailed and in-depth research on the Gene Therapy market. We also have centered on SWOT, PESTEL, along with different analysis of the industry.

Checkout FREE Report Sample of Gene Therapy Market Report for Better Recognizing: https://www.futuristicreports.com/request-sample/106731

(Kite Pharma Inc., Spark Therapeutics Inc., Novartis, GlaxoSmithKline PLC, Applied Genetic Technologies Corporation, NewLink Genetics Corp, Transgene SA, Oxford BioMedica, Genethon, Bluebird biInc.)

Youre able to thoroughly measure the competitions weaknesses and strengths together with our competitive analysis. The report, we have used total production and dispatch analysis in point of origin. Additionally, youre advised about the latest Gene Therapy industry advancements that will help you stay ahead of the competition. Our analysts are always on the feet to always track and analyze developments or changes in the Gene Therapy market. The analysis is full of statistical demonstrations and market statistics associated with sales, volume, CAGR, and share and regional and global predictions.

Gasoline Electric Solar

Rare Diseases Neurological Disorders Oncological Disorders Cardiovascular Diseases Infectious disease Other

The report supplies how big the Gene Therapy market will be in 2026 considering the studys base year 2019 and 2020. The market dynamics predominant in North America, Europe, Asia Pacific, Middle East, and Africa, and Latin America were taken into consideration and estimating the rise of the worldwide sector.

Key Questions Answered in this Report:

Futuristic Reports

Name: Alex CubbinsTel: +1-408-520-9037Email: [emailprotected]

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Gene Therapy Market Regions, Type and Application, Futuristic Study - Factory Gate

Merck is dipping its toes into a cell therapy partnership with A2 Biotherapeutics, with an offer to co-fund clinical development and allogeneic manufacturing activities through Phase I.

In particular, the pharma giant has its eyes on an undisclosed candidate utilizing A2s Tmod platform, which combines activation and blocking mechanisms in order to kill tumor cells while sparing healthy ones.

The deal features an upfront, an equity investment and reimbursement of certain expenses. Merck is also promising opt-in and milestone payments plus royalties, while keeping the door open for collaboration on a separate program.

A2 Biotherapeutics, which closed $71.5 million in Series B funding in October, has two other programs that are further along in the lead optimization phase.

The new pact brings Mercks immunotherapy and other expertise for the Tmod candidate especially in the later stages of development, manufacturing and commercialization and enables A2 to build allogeneic product development and manufacturing capabilities, said Scott Foraker, A2s president and CEO. Amber Tong

Small rare diseases biotech Soligenix $SNGX did not have a good Tuesday.

The Princeton, NJ-based company posted a Phase III fail in head and neck cancer, saying its SGX942 program did not produce a statistically significant outcome. Soligenix had been looking to treat severely inflamed mucous membranes resulting from other cancer therapies, but came up short.

Tuesdays news crushed the companys stock price, as shares were sliced by more than half at a 54% drop as the market opened. The price rebounded slightly by the end of the day, but still resulted in a 49% loss.

Soligenix did not report a p-value from the primary endpoint, which was the median duration of severe oral mucositis. They did note the data showed a 56% reduction compared to placebo, as the median in the control arm came in at 18 days and 8 days in the treatment arm.

The company tried to shine a light on a positive secondary endpoint 50% reduction in the duration of SOM in the per-protocol population. Soligenixs p-value came in here at 0.049, just clearing the statistically significant hurdle. The biotech said this endpoint may point to some evidence of biological activity.

The study enrolled 268 patients randomized 1:1 to receive either SGX942 or placebo. Soligenix said it will turn its attention toward another program, SGX301, in the treatment of cutaneous T cell lymphoma. Max Gelman

Tapping a new source for new gene therapy programs, BridgeBio has set up a three-year alliance with the University of California, San Francisco to identify early translational research that it can accelerate into the clinic.

BridgeBio, which is based in the Bay Area, said the deal follows a six-month pilot and is designed to formalize collaborative relationships with academic scientists.

The collaborations may initially take the form of sponsored research agreements with certain labs, it added, which may then lead to creation of new affiliate companies under the BridgeBio portfolio.

That pipeline currently lists three programs, utilizing AAV vectors to deliver corrective genes for congenital adrenal hyperplasia, Canavan disease and nonsyndromic hearing loss, respectively.

They are ready for more. Earlier this year, BridgeBio inked an agreement with Catalent to secure dedicated gene therapy development and manufacturing capacity to support its needs down the line. Amber Tong

Gritstone Oncology $GRTS has some new cash to play with.

The Emeryville, CA-based biotech announced Wednesday morning it had raised $110 million in private placement funding. Wednesdays deal valued company shares at $3.34 apiece, or Tuesdays closing price, and Gritstone said the funding would be primarily directed toward its GRANITE and SLATE pipeline candidates, two cancer immunotherapies.

News of the funding was met with cheers by investors, as Gritstones stock was up more than 13% in early Wednesday trading.

GRANITE, a personalized neoantigen-based immunotherapy, is being evaluated in combination studies in the Phase II portion of a Phase I/II study for microsatellite stable colorectal cancer. SLATE is also neoantigen-based and uses the same delivery system as GRANITE, but contains a fixed set of antigens rather than personalized. Its also being looked at in combination studies at the Phase II portion of a Phase I/II trial for NSCLC.

The financing was led by existing and new investors, including Redmile Group, Avidity Partners and EcoR1 Capital. The deal is expected to close by Dec. 28. Max Gelman

After closing its Series A round, Epsilon Molecular Engineering opened it back up, bagging 570 million ($5.5 million) total from investors, two bank loans and leasing.

The Saitama University spinout will use the funds for its work with heavy chain single domain antibodies, including collaborative research with Kitasato University and Kao Corporation on potential Covid-19 treatments.

EME aims to discover medium sized molecular bio-drugs with new modalities using its proprietary VHH technology, president Naoto Nemoto said in a statement. We will leverage this financing to accelerate collaborative research with pharmaceutical manufacturers and internal research using its own pipeline.

The round was led by Mitsubishi UFJ Capital, with help from Gunma Medical Engineering Vitalization Investments, Gunma Bank and Kao Corporation. EME also went forward with a subordinated loan from Shoko Chukin Bank, a loan from Saitama Resona Bank, and a lease from Syutoken Leasing. Nicole DeFeudis

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News briefing: Merck buys into A2's T cell therapy platform; Small Soligenix reports PhIII fail in head and neck cancer - Endpoints News

Last call for raising funds beforeChristmas, andthese companies didnt hesitate to scoop up their bags of cash.

TG Therapeutics

TG has been on the Nasdaq for over sevenyears now, but 2020 has been theyear for the New Yorkbiopharmato soar.With shares trading atless than $11 apiece in January, the stock is now well over $50 since theannouncementof positive topline results from two global Phase III trials for relapsing forms of MS.With a high probability of FDA approval, TG cashed inwith an upsized common stock offering,raising$300 millionto further develop and commercializetheir therapies.

BioAtla

This San Diego company hopped on the bandwagon of biopharma IPOs last week, offering 10.5M million shares at $18 apiece, a 38% increase, scooping up$189 millionin proceeds.BioAtlais developing a novel class of specific and selective antibody-based therapeutics.The companys conditionally active biologics only activate when they detect proximity to a tumor, thereby reducingsystemictoxicity.Funds will propel the companys two lead programs through three Phase II trials.

Cullinan Oncology

Breaking down the silos of drug research, Cullinan applies open innovation and collaboration to developa portfolio of first-in-class and best-in-classcancer therapies. With an oversubscribed$131.2 millionSeries C, the Cambridge company can advanceitsseven-candidatepipelineinto the clinic.Each candidate isstructured as a separate company managed by Cullinan. Two are currently in Phase I with an inhibitor drug for NSCLC and amonoclonal antibody reinvigorating the MICA/NKG2D axis.

Neurogene

New York-basedNeurogeneis establishing itself as a leader in gene therapies for neurological diseases. Last weeks$115 millionSeries B round willhelp advance multiple of the companys candidates into the clinic. The first of which targetslate infantileBattens Disease,a rare nervous system disorder that worsens over time and is fatal, usually 8-10 years old.The funds will also be used to build outNeurogenesadeno-associated virus vector GMP manufacturing capabilities.

Neuron23

Having worked undercover for two years, Neuron23 uncloakedlast weekwith$113.5 millionin financing for its launch.$30 million of the funds came from Westlake Village BioPartners, who just announcedtwo funds totaling$500 millionwith the intent to invest in Series A startups in the most promising companies.Neuron23 has hit the ground running, aiming to take on Parkinsons diseaseagainst giant Biogen, who recentlyorchestratedin a $1 billion dealwith Denaliwith the same target in mind. The plan is to start trials with healthy volunteers next year.

Neomorph

Established earlier this year, San Diego-based Neomorph raked in$109 millionin a Series A. The companys focus is on targeted proteindegradation, which offers opportunities for treatment developments across the board, including oncology.The Neomorph team has deep expertise in pharmacological approaches to targeted protein degradation and we are excited to be developing new therapeutics for patients with diseases that are currently difficult to treat, said scientific founderScottArmstrongMD, Professor of Pediatrics at Harvard Medical School and the Dana-Farber Cancer Institute.

AtsenaTherapeutics

Gene therapy startupAtsenaclosed on an oversubscribed$55 millionSeries A.The funds will be used to advance itsgene therapy for one of the most common cause of blindness in children through clinical trials.Leber congenital amaurosis (LCA) causes blindness in 2 to 3 out of 100,000 newborns.The company isalso planning for growth,looking to move into a larger space next yearto scale up gene therapy manufacturing. Ramping up across the board, theres a goal to hire 20 more positions with the move.

ONL Therapeutics

With support from Johnson & Johnson and more, Michigan-based ONL closed a$46.9 millionSeries B Preferred Stock financing round.The company is developing therapies for protecting the patients with retinal disease from vision loss.This funding supports the completion of a Phase 1 study in retinal detachment with ONLs lead compound ONL1204. In addition, the funding will advance ONL1204 in two chronic indications, glaucoma and dry age-related macular degeneration.

Peptilogics

Peptilogicsis the most recent biotech receiving investments fromPaypalsco-founder Peter Thiel. This week, Thiel participated in a$35 millionfinancing round for the Pennsylvania-basedcompany.The funds will be used to advancePeptilogicsproprietarycomputational peptide drug design and discovery platform. The platform discovers connections in diverse biomedical data and maps peptide sequences.

Octave Bioscience

Looking totakeitsfully integratedcare management platform to the next level, Octave completed a$32 millionSeries B financing round. The funds will allow thecompany to complete development of management products and services, expand clinical data and begin commercialization to neurologists and patients. Octaves first target has been multiple sclerosis patients, but will expand to other chronic, debilitating neurodegenerative diseases. Theplatformtracksblood-basedbiomarkers, enhanced MRI insights and mobile patient monitoring tools to feed intocare pathwaymodels to generatebetter patient outcomes and lower costs.

Vivace Therapeutics

Small molecule player Vivace closed a$30 millionSeries C for further development of its first-in-class therapies targeting the Hippo pathway. Funds will be usedto take its lead candidate into first-in-human studies in early 2021, targeting tumors dependent on activated YAP.Pre-clinical research has shown promise for the candidate both as a monotherapy and in combination with other anti-cancer therapies.

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Biopharma Money on the Move: December 16-22 - BioSpace

MALVERN, Pa., Dec. 23, 2020 (GLOBE NEWSWIRE) -- Ocugen, Inc., (NASDAQ: OCGN), a leading biopharmaceutical company focused on discovering, developing and commercializing a pipeline of innovative therapies, today announced the appointment of a vaccine scientific advisory board comprised of leading academic and industry experts in the vaccine field to evaluate the clinical and regulatory path to approval in the US market of Bharat Biotechs COVAXIN, a whole-virion inactivated COVID-19 vaccine candidateto be co-developed by Ocugen and Bharat Biotech for the US market.

Dr. Shankar Musunuri, Chairman, CEO, and Co-Founder of Ocugen remarked, We are thrilled to welcome this group of esteemed thought leaders to the Ocugen team to assist in our co-development with Bharat Biotech of COVAXIN. This unique yet traditional vaccine candidate is different from other options currently available in the US market with potentially broader coverage against multiple protein antigens of the virus.

The vaccine scientific advisory board consists of:

About Ocugen, Inc.Ocugen, Inc. is a biopharmaceutical company focused on discovering, developing, and commercializing transformative therapies to cure blindness diseases. Our breakthrough modifier gene therapy platform has the potential to treat multiple retinal diseases with one drug one to many and our novel biologic product candidate aims to offer better therapy to patients with underserved diseases such as wet age-related macular degeneration, diabetic macular edema, and diabetic retinopathy. For more information, please visit http://www.ocugen.com.

Cautionary Note on Forward-Looking StatementsThis press release contains forward-looking statements within the meaning of The Private Securities Litigation Reform Act of 1995, which are subject to risks and uncertainties. We may, in some cases, use terms such as predicts, believes, potential, proposed, continue, estimates, anticipates, expects, plans, intends, may, could, might, will, should or other words that convey uncertainty of future events or outcomes to identify these forward-looking statements. Such statements are subject to numerous important factors, risks and uncertainties that may cause actual events or results to differ materially from our current expectations. These and other risks and uncertainties are more fully described in our periodic filings with the Securities and Exchange Commission (the SEC), including the risk factors described in the section entitled Risk Factors in the quarterly and annual reports that we file with the SEC. Any forward-looking statements that we make in this press release speak only as of the date of this press release. Except as required by law, we assume no obligation to update forward-looking statements contained in this press release whether as a result of new information, future events or otherwise, after the date of this press release.

Ocugen Contact:Ocugen, Inc.Sanjay SubramanianChief Financial Officerir@ocugen.com

Media Contact:LaVoieHealthScienceLisa DeScenzaldescenza@lavoiehealthscience.com+1 978-395-5970

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Ocugen Establishes Vaccine Scientific Advisory BoardLeading experts to evaluate the clinical and regulatory path to approval in the US market of...

Advances Eden BioCells clinical program to validate Rapid Personalized Manufacturing (RPM)

Clinical trial to study autologous CD19-specific CAR-T using RPM technology designed to reduce cost and simplify production for infusion the day after gene transfer

BOSTON, Dec. 21, 2020 (GLOBE NEWSWIRE) -- Ziopharm Oncology, Inc. (Ziopharm or the Company) (Nasdaq: ZIOP), today announced that the Taiwan Food and Drug Administration has cleared an investigational new drug application (IND) from Eden BioCell, a joint venture between Ziopharm and cell therapy company TriArm Therapeutics, for its phase 1 clinical trial to evaluate patient-derived CD19-specific CAR-T, using Ziopharms Rapid Personalized Manufacturing (RPM) technology. This is an investigational treatment for patients with relapsed CD19+ leukemias and lymphomas and the first clinical study of autologous non-viral CD19-specific CAR-T in Taiwan.

This trial will utilize Ziopharms non-viral Sleeping Beauty cell engineering technology to infuse autologous CAR-T the day after T cells have been genetically modified. Ziopharms RPM CD19-specific CAR-T therapy results from the stable, non-viral insertion of DNA into the genome of resting T cells to co-express the chimeric antigen receptor (CAR), membrane-bound IL-15 (mbIL15) and a safety switch. The trial is being conducted at National Taiwan University Hospital.

This study is a testament to the relationship Ziopharm has quickly established with Eden BioCell and TriArm and the progress using patients T cells under RPM to target malignancies, said Laurence Cooper, M.D., Ph.D., Chief Executive Officer of Ziopharm. The results will help us understand the benefit of engineering T cells with membrane bound IL-15 which could benefit not only CAR-T, but also the engineering of T cells to express T-cell receptors.

Jay Zhang, Co-Founder and Chief Executive Officer of TriArm, added, We are very excited to receive clearance of our IND in Taiwan. The learnings from this study will build upon the encouraging early data we are seeing with patients treated with RPM CAR-T targeting CD19 malignancies under compassionate use. We believe our approach has the potential to transform CAR-T therapy by dramatically decreasing the amount of time needed for manufacturing engineered T cells, thereby increasing efficacy and decreasing cost.

CAR-T therapy has proved an effective therapy for B-cell cancers, noted Dr. Shang-Ju Wu, Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital and Principal Investigator for the study. Further optimization by shortening the manufacturing time would be of great importance to make this therapy more available to patients. We are honored to be involved in the clinical development of this non-viral CAR-T therapy produced using RPM. We hope the data derived from this current trial will advance CAR-T therapy to benefit our patients.

Up to 24 patients with relapsed CD19+ leukemias and lymphomas will be enrolled in this phase 1 trial, with the goal of infusing 16 subjects (Taiwan FDA #1096030182). The primary endpoint of the study is to evaluate the safety and tolerability of autologous CD19-specific T cells manufactured using the RPM process.

About Eden BioCell In December 2018, Ziopharm and TriArm Therapeutics announced the launch of Eden BioCell to lead clinical development and commercialization of Sleeping Beauty-generated CAR-T therapies in Greater China. Ziopharm licensed the rights to Sleeping Beauty-generated CAR-T therapies targeting the CD19 antigen using Ziopharms RPM technology in Greater China to Eden BioCell. TriArm has committed up to $35 million to this joint venture, and Eden BioCell is owned 50-50 by Ziopharm and TriArm.

About TriArm TherapeuticsTriArm Therapeutics is a cell therapy company formed by Panacea Venture with R&D operations in Germany, United States and Greater China region. The company is dedicated to the treatment of cancer and autoimmune diseases.

About Ziopharm Oncology, Inc.Ziopharm is developing non-viral and cytokine-driven cell and gene therapies that weaponize the bodys immune system to treat the millions of people globally diagnosed with cancer each year. With its multiplatform approach, Ziopharm is at the forefront of immuno-oncology. Ziopharms pipeline is built for commercially scalable, cost effective T-cell receptor T-cell therapies based on its non-viral Sleeping Beauty gene transfer platform, a precisely controlled IL-12 gene therapy, and rapidly manufactured Sleeping Beauty-enabled CD19-specific CAR-T program. The Company has clinical and strategic collaborations with the National Cancer Institute, The University of Texas MD Anderson Cancer Center and Regeneron Pharmaceuticals. For more information, please visit http://www.ziopharm.com.

Forward-Looking Statements DisclaimerThis press release contains forward-looking statements as defined in the Private Securities Litigation Reform Act of 1995, as amended. Forward-looking statements are statements that are not historical facts, and in some cases can be identified by terms such as "may," "will," "could," "expects," "plans," "anticipates," and "believes." These statements include, but are not limited to, statements regarding the potential benefits of the Companys CAR-T therapy and the Companys expectations regarding the number of patients expected in this phase 1 clinical trial. Although Ziopharms management team believes that the expectations reflected in such forward-looking statements are reasonable, investors are cautioned that forward-looking information and statements are subject to various risks and uncertainties, many of which are difficult to predict and generally beyond the control of Ziopharm, that could cause actual results and developments to differ materially from those expressed in, or implied or projected by, the forward-looking information and statements. These risks and uncertainties include among other things, changes in Eden BioCells operating plans that may impact its cash expenditures, the uncertainties inherent in research and development, future clinical data and analysis, including whether any of Ziopharms product candidates will advance further in the preclinical research or clinical trial process, including receiving clearance from the U.S. Food and Drug Administration or equivalent foreign regulatory agencies to conduct clinical trials and whether and when, if at all, they will receive final approval from the U.S. FDA or equivalent foreign regulatory agencies and for which indication; the strength and enforceability of Ziopharms intellectual property rights; competition from other pharmaceutical and biotechnology companies as well as risk factors discussed or identified in the public filings with the Securities and Exchange Commission made by Ziopharm, including those risks and uncertainties listed in Ziopharms Quarterly Report on Form 10-Q filed by Ziopharm with the Securities and Exchange Commission. We are providing this information as of the date of this press release, and Ziopharm does not undertake any obligation to update or revise the information contained in this press release whether as a result of new information, future events or any other reason.

Investor Relations Contacts:Adam D. Levy, PhD, MBAEVP, Investor Relations and Corporate CommunicationsT: 508.552.9255E: alevy@ziopharm.com

Media Relations Contact:LifeSci Communications:Patrick BurseyT: 646.876.4932E: pbursey@lifescicomms.com

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Ziopharm Oncology Announces Clearance of Taiwan's First IND of Non-viral CAR-T for the Treatment of Relapsed CD19+ Leukemias and Lymphomas -...