For some people with blood cancers who need a stem cell transplant, finding a donor who is an excellent match can mean the difference between life and death.

Unfortunately, even though there are more than 40 million potential donors in the national registry, finding a perfect match isnt always possible, especially in underrepresented racial and ethnic groups.

But a new approach using an old chemotherapy drug,cyclophosphamide, isis opening up new possibilities for people with cancers like leukemia, lymphoma, and multiple myeloma. Researchers have found that by administering the drug several days after transplantation, people receiving blood stem cells from unrelated, partially matched donors can have survival rates comparable with those who received exactly matched cells.

[1]

This innovative approach can greatly expand patient access to safe and effective stem cell transplant, regardless of matching degree with the donor, says lead coauthor Monzr M. Al Malki, MD, a hematologist and oncologist and director of the Unrelated Donor BMT program at City of Hope, a cancer research and treatment organization with locations across the United States.

Thats exciting because it means more patients will be able to receive this potentially life-extending therapy, says Dr. Al Malki.

Donor compatibility is determined by a set of protein markers on blood cells called HLAs (human leukocyte antigens), says David Miklos, MD, a professor of medicine and chief of Stanford BMT and Cell Therapy Program at Stanford Medicine in California. Stanford was one of the medical sites of the trial, though Dr. Miklos is not a coauthor of the research.

[2]

[3]

Why was an exact match needed? Anything less increased the likelihood of a graft failure, as well as graft-versus-host disease meaning the transplanted cells attack the patients own, which can cause serious or even fatal complications, explains Miklos.

About a decade ago, researchers started using cyclophosphamide to destroy the parts of a persons immune system that would reject the transplant. That breakthrough allowed researchers to not only have better outcomes in fully matched donors, it also opened the door for successful transplants between people who were only partial matches.

[4]

The new study looked at cyclophosphamide treatment in patients receiving peripheral blood stem cell transplantation meaning healthy stem cells are harvested from a donors bloodstream, and then administered via infusion to the person with cancer.

Blood stem cell transplantation has largely replaced bone marrow transplantation, according to researchers.It's an easier way of collecting stem cells from donors, and its a little safer, because donors dont need to be under anesthesia as they would in bone marrow transplantation, says Al Malki.

For this part of the study, the researchers examined data from 70 adults who were 65 years old on average, all with advanced blood cancers. Participants received a reduced-intensity conditioning regimen to somewhat suppress their immune system to prepare them for transplantation, followed by an infusion stem cells from unrelated, partially matched donors.

The researchers reported an overall high survival rate of 79 percent at one year which is comparable to survival rates seen with fully matched donors.

The main side effect or risk of transplantation is graft-versus-host disease, says Al Malki. After one year, 51 percent of participants were free of the disease and had not relapsed, which is also comparable to what would be seen with fully matched donors, he says.

Historically, barriers in access to transplant have existed due to the low availability of matched, related sibling donors, as well as the substantial variance of matched, unrelated donor availability, especially for patients with diverse ancestry, says study coauthor Steven M. Devine, MD, chief medical officer of NMDP (formerly known as the National Marrow Donor Program and Be The Match).

These findings advance our ability to offer more options to patients without a fully matched donor, many of whom are ethnically diverse and have been underserved in receiving potentially lifesaving cell therapy, says Dr. Devine.

These findings are incredibly important and critical in the effort to improve existing inequities, says Miklos.

In the past, we could not bring some patients forward to receive this lifesaving therapy because they didnt have a compatible donor, but with the new approach of using post-transplant cyclophosphamide, all patients have donors now, he says.

See the rest here:
Cancer Patients Who Need Stem Cell Transplants May Have New Donor Options - Everyday Health

Recommendation and review posted by Bethany Smith

A potential therapy for Alzheimer's disease highlights the important role of the immune system and how stem cell transplants can help revitalize aging cells linked to the neurodegenerative disease in mice.

(CN) Scientists on Tuesday revealed how young bone marrow stem cell transplants could be the future of treating immune cells that assist with the progression of Alzheimers Disease.

The findings in Science Advances come from a team of Chinese researchers who successfully transplanted bone marrow from two-month-old mice to restore the immune systems of their older counterparts affected by genetic and pathological indicators of Alzheimers disease.

Alzheimers is a progressive and fatal neurological disease that commonly affects people over the age of 65. Prevailing Alzheimers research links the disease to the buildup of amyloid beta and tau proteins in the brain, though other recent studies have found that approximately half of the genes associated with Alzheimer's are directly involved with the immune system.

As the researchers explain in the study, aged immune systems undergo immunosenescence the gradual decline of immune cell production and functionality which partly derives from aging bone marrow hematopoietic stem cells and progenitor cells.

Progenitor cells develop or differentiate into a predetermined type of cell. Hematopoietic stem cells are the source of peripheral immune cells like monocytes, macrophages and dendritic cells all of which can assist in the progression of neurodegenerative and neuroinflammatory diseases when they reach the brain.

The process of immunosenescence thus drives systemic aging and contributes to an increased susceptibility to age-related diseases like Alzheimer's disease. Using this information, the team realized that replenishing bone marrow with young hematopoietic stem cells can rejuvenate older immune cells and intervene in Alzheimers symptoms.

And thats not all they found.

Our findings revealed that aging induced changes in the gene expression in both innate and adaptive immune cells, aligned with the dysfunction of both innate and adaptive immune responses observed in aging animals or elderly individuals, such as diminished phagocytosis function of monocytes, impaired antiviral immunity of [natural killer] cells, elevated production of autoantibodies by B cells and expansion of cytotoxic T cells, the authors wrote.

The team also found that the genetic markers associated with aging were enriched for Alzheimers-related pathways, indicating an active link between senescent or deteriorating peripheral immune cells and the development of Alzheimer's especially monocytes.

Monocytes are a type of white blood cell that can clear amyloid beta proteins in the brain and plasma through phagocytosis (kind of like a cellular version of Pac-Man). Aging, however, can impair this ability and accelerate the occurrence of Alzheimers.

The findings from this study suggest that the diminished monocytic A clearance capacity is a consequence of the downregulation of key receptors involved in A phagocytosis within aging monocytes, the authors wrote. Collectively, these pieces of evidence indicate that the senescence of peripheral immune cells plays a critical role in the pathogenesis of AD, and rejuvenating peripheral immune cells in aging individuals may represent a promising intervention strategy.

As for the therapeutic value of young bone marrow transplants, the authors say there are many.

Not only did the young bone marrow improve the physical and behavioral symptoms of Alzheimers in older mice, but it decreased amyloid beta levels, lowered cerebral amyloid beta plaque and improved the mices overall cognition.

The findings also suggest that transplants can reverse one-third of Alzheimers-related gene expression, alleviate aged pathways, restore altered cell-to-cell communication in aging peripheral blood mononuclear cells, rescue dysfunctional monocytic functions and reduce levels of secreted triggers from aging cells called blood senescence-associated secretory phenotype or SASP factors.

The authors added that since young bone marrow transplants enhance the overall phagocytosis of monocytes, the same intervention is worth exploring for mice targeted by tau proteins, the other hallmark of Alzheimers. Future studies, they wrote, could focus on exploring other new strategies that can rejuvenate immune cells to advance the possibility of clinical translation.

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Stem cell transplants deliver promising treatments for mice with Alzheimer's disease - Courthouse News Service

Recommendation and review posted by Bethany Smith

Study selection

From the initial literature search, we retrieved relevant 3948 records from PubMed, Web of Science, Scopus, and the Cochrane Library. After the title and abstract screening of them we screened the full text of 295 articles. Only nine studies met our criteria31,32,33,34,35,36,37,38,39. Figure1 shows the PRISMA flow diagram of our search and selection process.

The nine studies were RCTs and enrolled a total of 422 multiple sclerosis patients. All studies were parallel in design except 4 studies were cross-over RCTs31,32,34,39. These cross-over trials were reviewed up to the point of cross-over. All studies infused stem cells intravenously except Petrou et al. that included an additional intrathecal SCT subgroup32. This study showed that intrathecal SCT was more effective than intravenous SCT, but we pooled the data of both routes as single study data. Of the included studies, only two studies used autologous hematopoietic SCT (AHSCT) in addition to immune ablative regimen prior to the transplantation37,38. Burt et al. compared SCT to DMTs (natalizumab, fingolimod, and dimethyl fumarate) in RRMS patients37, and Mancardi et al., compared SCT to mitoxantrone in relapsing and progressive MS patients38.

Supplementary Table S1 summarizes the characteristics of the included trials, Table 1 shows the demographic and baseline characteristics of these studies population, and Supplementary Table S3 shows efficacy endpoints reported at 6months.

We assessed seven domains in each study according to The Cochrane Collaborations tool for assessing risk of bias 1. The 9 studies were randomized but 4 studies32,35,38,39 didnt clarify the methods of random sequence generation. 6 RCTs confirmed concealment of patients allocation to the intervention31,32,33,34,36,37. Blinding of the outcome assessors was clearly stated in all studies except Nabavi et al.39 but blinding of participants and personnel wasnt fulfilled in three studies35,37,38. The reasons for incomplete outcome data are related to the treatment in Uccelli et al.31 and the reasons werent clearly described in Burt et al37. Two studies reported the outcomes in an incomplete way that limited their inclusion in the meta-analysis inducing a reporting bias33,38. The overall quality of the studies was good for 2 studies32,36, fair for 3 studies31,34,37, and poor for 4 studies33,35,38,39. Figure2 shows the risk of bias summary and graph.

Risk of bias assessment: (a) Risk of bias summary, (b) Risk of bias graph.

After analyzing the efficacy and safety outcomes for all studies collectively, we subdivided the results into studies that used immunosuppression before AHSCT37,38 and studies that transplanted mesenchymal stem cells (MSCs) without immunosuppression31,32,33,34,35,36,39 to minimize the procedural variations among the included trials.

The majority of the studies31,32,33,34,35,36,37,39 reported EDSS change for 211 patients in stem cell transplantation (SCT) arm and 176 controls. Because the time of reporting this outcome varied among the studies, we analyzed EDSS change at the last follow-up reported by each study. Our analysis showed nonsignificant difference between SCT group and the control group (MD=0.48, 95% CI [1.11, 0.14], p=0.13). There was great heterogeneity between studies (2=116.74, df=7, p<0.00001, I2=94%), so we pooled the data under the random-effects model (Table 2 and Supplementary Figure S1).

The subgroup analysis of the studies that used MSCs without immunosuppression also showed nonsignificant improvement (MD=0.3, 95% CI [0.87, 0.27], p=0.3). However, Burt et al37. that used immunosuppression before AHSCT revealed significant EDSS reduction (Supplementary Figure S1).

The results remained nonsignificant after the leave-one-out sensitivity analysis (Supplementary Figure L1).

The heterogeneity within the studies was not significant (2=1.61, df=1, p=0.2, I2=38%), and we adopted a random effect model. The reduction of EDSS in SCT group was significantly greater than the control group (MD=0.57, 95% CI [1.08, 0.06], p=0.03) (Table 2 and Fig.3a).

Forest plot of EDSS change from baseline at (a) 2months, (b) 6months, (c) 12months.

Adopting the random-effects model, the heterogeneity between the studies was significant (2=65.27, df=6, p<0.00001, I2=91%), and SCT showed nonsignificant improvement of EDSS compared to the control (MD=0.48, 95% CI [0.98, 0.03], p=0.07) (Table 2 and Fig.3b). MSCs without immunosuppression also resulted in nonsignificant EDSS reduction at 6months (MD=0.33, 95% CI [0.78, 0.11], p=0.14) (Fig.3b).

The effect estimate changed to (MD=0.62,95% CI [1.14, 0.09], p=0.02) favoring SCT over the control after excluding Nabavi et al.39 from the analysis (Supplementary Figure L2 and Table L1).

We adopted the random-effects model because heterogeneity was significant, and the difference between the SCT group and the control was not significant at 12months for both collective studies analysis and studies used MSCs without immunosuppression (p=0.06 and p=0.5, respectively). However, the study that used AHSCT plus immunosuppression37 showed significant improvement in patients disability (p<0.00001) (Table 2 and Fig.3c).

After performing a sensitivity analysis by excluding Fernandez et al.36, the results changed from nonsignificant to significant improvement in SCT arm (MD=1.69, 95% CI [1.94, 1.44], p<0.00001) (Supplementary Figure L3 and Table L1).

We compared the effect of SCT on patients disability depending on baseline EDSS. Six studies31,32,33,34,37,39 included 334 MS patients with baseline EDSS6.5, while two studies35,36 included 53 patients with baseline EDSS>6.5. Using a random effects model, both subgroups showed significant heterogeneity (p<0.00001 and p<0.00001). Both subgroups revealed nonsignificant effect of SCT on EDSS, (MD=0.41, 95% CI [1.11, 0.29], p=0.25) for baseline EDSS6.5 subgroup and (MD=0.68, 95%CI [2.68, 1.32], p=0.5) for baseline EDSS>6.5 subgroup (Table 2 and Supplementary Figure S2).

We pooled data of EDSS change from baseline to the last assessment time under a random-effects model, and the differences were nonsignificant for both low and high doses subgroups, (MD=0.31, 95% CI [1, 0.38], p=0.37) and (MD=0.57, 95% CI [1.94, 0.8], p=0.41), respectively. The studies of both subgroups showed significant heterogeneity (I2=95%, p<0.00001) for the low doses subgroup, and (I2=89%, p=0.0001) for the high doses subgroup (Table 2 and Supplementary Figure S3).

Adopting a random-effects model, stem cells from embryonic as well as adult origin showed nonsignificant effect on EDSS (p=0.17, and p=0.37, respectively), With significant heterogeneity among the studies (I2=88%, p=0.004), and (I2=94%, p<0.00001), respectively (Table 2 and Supplementary Figure S4).

We pooled data using a random-effects model. Five studies31,32,33,36,39, in which placebo was the control, showed substantialheterogeneity (I2=63%, p=0.03) and the difference between SCT and placebo was not significant (MD=0.09, 95% CI [0.46, 0.28], p=0.62). Three studies34,35,37, in which the control was active treatment, showed significant reduction of EDSS with SCT compared to the active drugs (MD=1.21, 95% CI [1.98, 0.43], p=0.002) and the heterogeneity was significant (I2=88%, p=0.0002) (Table 2 and Supplementary Figure S5).

Only two studies32,34 reported the number of relapses in the 6months following the intervention. Under a random-effects model, the heterogeneity was moderate (p=0.14, I2=53%), and the decrease in relapses number was nonsignificant (p=0.23) (Supplementary Figure S6).

Four studies31,32,34,37 assessed T25-FW in 154 and 136 patients in the SCT and control groups, respectively. We pooled data under a random-effect model, and heterogeneity was moderate (2=5.99, df=3, p=0.11, I2=50%). SCT resulted in a nonsignificant improvement in patients T25-FW scores compared to the control group (MD=0.69, 95% CI [1.93, 0.56], p=0.28), as shown in Fig.4.

Forest plot of T25-FW change from baseline.

In the studies that included mesenchymal SCT without immunosuppression, the improvement in patients T25-FW scores after SCT was not significant (MD=0.39, 95% CI [0.84, 0.06], p=0.09), but T25-FW significantly improved in the study that used AHSCT and immunosuppression37 (p=0.006). Figure4 demonstrates these analyses. The p value of the results didnt change after the one-study-removed sensitivity analysis (Supplementary Figure L4).

9-HPT was evaluated in four RCTs31,32,34,37. We used a random-effects model because heterogeneity was significant (p=0.0003, I2=84%). 9-HPT showed nonsignificant improvement in the collective analysis and the sub-analysis of MSCs without immunosuppression. However, Burt et al.37. revealed a significant improvement (p<0.00001) (Supplementary Figure S7). The results remained nonsignificant after sensitivity analysis (Supplementary Figure L5).

We pooled PASAT-3 scores assessed at the end of treatment in four trials under a random-effects model31,34,36,37. Heterogeneity was minimal (p=0.35, I2=9%), and the differences were nonsignificant in the collective analysis and the sub-analysis of autologous and mesenchymal SCT (p=0.35, p=0.96, and p=0.31, respectively) (Supplementary Figure S8). Effect estimate remained nonsignificant after one-study-removed sensitivity analysis (Supplementary Figure L6).

We analyzed the change in brain lesion volume from baseline to the end of the follow-up. Data were pooled under a random-effects model, heterogeneity was absent (p=0.38, I2=0%). Our analysis revealed a significant reduction in T2 lesions volume (MD=7.05, 95% CI [10.69, 3.4], p=0.0002). In the studies that used MSCs without immunosuppression, the reduction of brain lesions volume was nonsignificant (p=0.1) (Fig.5a).

Forest plot of radiological outcomes change from baseline (a) MRI T2-weighted lesions volume at the end of treatment, (b) MRI T2-weighted lesions number at 12months, (c) number of GELs at the end of treatment. *the study used immunosuppression before AHSCT.

The results became nonsignificant and changed to (MD=4.41, 95% CI [9.66, 0.85], p=0.1) after a sensitivity analysis performed by excluding Burt et al.37 (Supplementary Figure L7 and Table L1).

Adopting a random-effects model, the studies showed substantial heterogeneity (p=0.07, I2=70%). And the differences between SCT and the control after 12months were nonsignificant (p=0.99) (Fig.5b).

Five studies31,32,33,34,36 assessed this outcome. Four studies reported the change of GELs number from baseline at 6months except Fernandez et al.36 at 12months. We pooled data under a random-effects model and heterogeneity was not significant (2=7.81, df=4, p=0.1, I2=49%). Our analysis revealed nonsignificant differences in GELs number change (p=0.83) (Fig.5c). The results didnt change after sensitivity analysis (Supplementary Figure L8).

Seven studies31,32,33,34,36,37,38 reported adverse events that occurred during the follow-up period. Two studies35,39 didnt provide data about AEs. Nabavi et al. mentioned only pain at the site of bone marrow aspiration39. Our analysis revealed that the difference was nonsignificant between SCT and the control group regarding the incidence of most AEs. Administration-related AEs, including infusion site swelling, hematoma, and pain, were significantly more common in the SCT group compared to the control (N=25, RR=2.55, 95% CI [1.08, 6.03], p=0.034). On the other hand, the SCT group had a lower incidence of total infections (any infection during the follow-up period, including viral infections, respiratory, urinary infections, scabies, and other infestations) than the control group (N=60, RR=0.58, 95% CI [0.37, 0.9], p=0.02). Regarding the use of immunosuppression, AHSCT combined with immunosuppression was significantly associated with a higher incidence of blood and lymphatic system disorders (N=16, RR=2.33, 95% CI [1.23, 4.39], p=0.009). The analyses of the adverse events are shown in Table 3 and Supplementary Figures S9S14. No transplant-related mortality was noted in all trials during the follow-up period, except for two unrelated deaths compacted by Fernandez et al. in the placebo arm (one due to choking while feeding and the other due to respiratory infection)36.

We examined the publication bias among the studies that reported the effect of SCT on patients disability using the funnel plot test. Although there was funnel plot asymmetry, the test isnt reliable because the included studies were less than ten studies24 (Supplementary Figure S15).

Read the original here:
Efficacy and safety of stem cell transplantation for multiple sclerosis: a systematic review and meta-analysis of ... - Nature.com

Recommendation and review posted by Bethany Smith

In a recent study published in The New England Journal of Medicine, researchers developed an innovative all-in-one treatment protocol that combines CD7 chimeric antigen receptor T-cell therapy with subsequent haploidentical hematopoietic stem cell transplantation. This approach significantly advances the treatment of CD7+ hematological malignancies by eliminating the need for myeloablative chemotherapy and immunosuppressants for graft-versus-host disease prophylaxis.

Chimeric antigen receptor (CAR) T-cell therapy utilizes synthetic antigen receptors targeting T lymphocytes to attack tumor cells specifically. To date, CAR T-cell therapies have shown unprecedented efficacy in B-cell malignancies. In contrast, there are significantly fewer clinical trials of CAR T-cell therapy against T-cell malignancies.1 Outcomes of T-cell lymphomas and relapsed T-cell acute lymphoblastic leukemia (T-ALL) patients are notably poor compared to those of their B-cell counterparts, with an estimated 5-year overall survival rate of only 32% for T-cell lymphomas patients and 7% for relapsed T-ALL patients.2 The challenge in treating T-cell malignancies lies in the lack of ideal target antigens. Several studies, including a recent study published in Cell Research,3 identified CD7 as a promising target for CAR-T therapy in the treatment of T-cell malignancies. However, in these clinical trials of CD7 CAR T-cells, researchers frequently observed that CD7 CAR T-cell treatment resulted in incomplete hematologic recovery and pancytopenia, presenting a significant clinical challenge.4

Recently, The New England Journal of Medicine reported on a novel all-in-one treatment regimen developed by a team from Hangzhou, China (hereafter referred to as the Hangzhou Protocol).5 In this clinical trial of CD7 CAR T-cell therapy for CD7+ hematological malignancies, including T-cell malignancies and acute myeloid leukemia (AML), Hu et al. observed that CD7 CAR T-cell treatment induced severe bone marrow hypocellularity, pancytopenia, and immunosuppression, aligning with previous reports. The first patient experienced persistent grade 4 pancytopenia for three months following CAR T-cell infusion and consequently developed severe infections. A salvage haploidentical hematopoietic stem cell transplantation (HSCT) was thus performed, without additional pharmacologic pre-HSCT conditioning regimens. Favorable engraftment of hematopoietic stem cells (HSCs) and immune reconstitution were observed, suggesting that the potent immunosuppressive functions of CD7 CAR T-cells, coupled with prior lymphodepletion, might replace the need for traditional chemotherapy conditioning. The subsequent 9 patients of similar conditions were promptly subjected to HSCT within one month after receiving CD7 CAR T-cell therapy without chemotherapy conditioning and immunosuppressive drugs for graft-versus-host disease (GVHD) prophylaxis. All 10 patients treated with the Hangzhou Protocol achieved complete remission. Pancytopenia was successfully relieved after allogeneic HSCT. Eight patients had full donor chimerism and immune reconstitution with a mild and manageable incidence of acute GVHD. Six of these patients remained in minimal residual disease-negative complete remission. The one-year overall survival rate increased to 68%. Collectively, this all-in-one Hangzhou Protocol offers a promising therapeutic option to CD7+ tumor patients.

Traditional CAR T-cell therapy followed by bridging HSCT involves longer intervals, chemotherapy conditioning, and the use of immunosuppressants for GVHD prophylaxis. Notably, the Hangzhou Protocol offers quadruple clinical benefits (Fig.1). First, CD7 CAR T-cell therapy efficiently eliminates CD7+ cancer cells in patients. More than 95% of patients with T-cell malignancies have CD7+ tumor cells,6 and about 30% of AML patients exhibit CD7 expression.7 In the trial, all treated patients including those with both T-cell malignancies and AML achieved complete remission. Second, the Protocol eliminates the need for pharmacologic myeloablation before HSCT, thereby avoiding its associated toxic effects. This is particularly beneficial for patients who have severe physiological issues or are in poor condition and who often are ineligible for allogeneic HSCT. Third, the Hangzhou Protocol does not require the use of immunosuppressants for GVHD prophylaxis, thus avoiding subsequent immunodeficiency issues. This allows the patients immune system to recover more rapidly. However, the mechanism behind this benefit requires further investigation. It is reported that CD7 plays a costimulatory role in T-cell signaling.8 Therefore, CD7 T cells, which are generated post CD7 CAR T-cell treatment, display reduced functionality and consequently a lower degree of GVHD. Lastly, the all-in-one treatment regimen maintains the persistence of CAR T-cells and supports their long-term immune surveillance function along with the graft-versus-leukemia effect. This maximizes the benefits of long-term immune surveillance by CAR T-cells and minimizes the risk of tumor relapse.

The figure illustrates clinical benefits of the Hangzhou Protocol in the context of CD7 CAR T-cell therapy for hematological malignancies. The top left section highlights the targeted tumor clearance capability of CD7 CAR T-cells, which are effective against both T-cell malignancies and AML. The top right section demonstrates the elimination of the need for myeloablative chemotherapy, reducing patient exposure to toxic effects. The bottom left section shows the avoidance of immunosuppressants in GVHD prophylaxis, which not only facilitates quicker immune system recovery but also minimizes complications from immunosuppression and associated drug side effects. Finally, the bottom right section details the sustained CAR T and GVL effects, which are crucial for preventing tumor relapse and supporting long-term immune surveillance. GVL graft-versus-leukemia, BM bone marrow, GVHD graft-versus-host disease, AML acute myeloid leukemia, CAR chimeric antigen receptor.

Several questions remain to be addressed regarding CD7 CAR T-cell therapy. One interesting question is why CD7 CAR T-cells suppress the patients hematopoiesis, causing severe pancytopenia, bone marrow aplasia, and immunosuppression. CAR T-cell-induced cytokine release syndrome could induce cytokine-associated pancytopenia.9 Conversely, it is also possible that minimal expression of CD7 on HSCs might induce CAR T-cell cytotoxicity. Furthermore, the mechanisms behind the successful engraftment and immune reconstitution of donor-derived stem cells in the presence of CD7 CAR T-cells while the patients own hematopoiesis is suppressed, are intriguing and warrant further investigation. Another interesting point is why CD7 CAR T-cell persistence is significantly longer than that of CD19 CAR T-cells. CAR tonic signaling has been reported to play a crucial role in regulating in vivo persistence and fitness of CAR T-cells.10,11 Thus, it is necessary to explore whether CD7 knockout in CD7 CAR T-cells affects CAR tonic signaling. All these questions need to be addressed in future studies to enhance our understanding and improve the efficacy of CD7 CAR T-cell therapy.

More here:
All-in-one Hangzhou Protocol: killing four birds with one stone | Cell Research - Nature.com

Recommendation and review posted by Bethany Smith

Abu Dhabi Stem Cells Center (ADSCC) has received the internationally recognised The Foundation for the Accreditation of Cellular Therapy (FACT) accreditation for its comprehensive cellular therapy programme.

Under the leadership of its Chief Executive Officer Prof. Yendry Ventura, ADSCC is the first healthcare institution in the UAE to be recognised by FACT, positioning the UAE as a leading destination for cutting-edge medical advancements in the region.

Out of the 261 centres worldwide accredited by FACT for cellular therapy, ADSCC stands as one of only two centres in the entire Middle East to meet FACTs rigorous standards. This accomplishment solidifies ADSCC's position as a leader in advanced cellular therapy. The centre achieved FACT accreditation for FACT-JACIE International Standards for Hematopoietic Cellular Therapy Product Processing, which implement best quality standards of medical and laboratory practice in hematopoietic progenitor cell transplantation and therapies from any hematopoietic tissue source (marrow, peripheral blood, umbilical cord and placental blood).

In 2020, ADSCC launched its Abu Dhabi Bone Marrow Transplant programme, the first comprehensive programme to provide autologous and allogeneic hematopoietic stem cells transplant (HSCT) for adult and paediatric patients in the UAE. The programme is led by a team of top-notch hematologists and clinicians, offering advanced holistic care for patients with hematological diseases, in addition to genetic and autoimmune disorders. Since the launch of ADSCC in 2018, the centre has achieved various milestones such as manufacturing the first CAR-T Cell Therapy in UAE. It has also received multiple prestigious accreditations including its recognition as a Centre of Excellence in Hematopoietic Stem Cell Transplantation by the Department of Health - Abu Dhabi in 2023.

Prof. Yendry Ventura, CEO of ADSCC and Adjunct Professor at UAE University, said: Under the guidance of UAEs wise leadership and in line with the UAEs national agenda in healthcare, ADSCC has a clear vision and mandate of pioneering cellular therapy on a global scale and bringing medical breakthroughs closer to bedside, which plays a crucial role in positioning Abu Dhabi and the UAE at the vanguard of healthcare innovation. With this landmark FACT accreditation, we are breaking new ground in addressing complex diseases through cutting-edge treatments such as stem cell transplants and other cellular therapies. The journey towards the accreditation has been both extensive and stimulating, and we take immense pride in being the first institution in UAE to receive it. This achievement reaffirms our commitment to continue investing in providing the highest standards of patient care and offering top-tier cellular therapy services that is on par with the best globally.

Dr Phyllis Warkentin, FACT Chief Medical Officer, said: FACT accreditation represents a commitment to quality throughout an organisation and requires dedication and perseverance. I congratulate Prof. Ventura and the team of the Abu Dhabi Stem Cells Centre on achieving FACT accreditation, the first cellular therapy product processing laboratory in the UAE to reach this milestone.

The FACT accreditation programme conducts rigorous and comprehensive inspections awarding accreditation objectively based on evidence and in accordance with international standards and best practices in cellular therapies and is considered the leading accreditation body for cellular therapy programs worldwide.

Excerpt from:
Abu Dhabi Stem Cells Center first healthcare institution in UAE to receive FACT accreditation for cellular therapy ... - Abu Dhabi Media Office

Recommendation and review posted by Bethany Smith

Dublin, May 31, 2024 (GLOBE NEWSWIRE) -- The "North America Hormone Replacement Therapy Market Report by Product, Route of Administration, Type of Disease, and Country 2024-2032" report has been added to ResearchAndMarkets.com's offering.

The North America hormone replacement therapy market size reached US$ 4.5 Billion in 2023. Looking forward, the market to reach US$ 6.9 Billion by 2032, exhibiting a growth rate (CAGR) of 4.9% during 2023-2032

This treatment is particularly favorable for patients who are experiencing growth hormone deficiency, women nearing menopause and older people suffering from hypogonadism. HRT is available in several forms such as gels, injections, implants, and skin and mouth patches (transdermal). However, it may not be suitable for patients that have a record of blood clots, liver disease and untreated high blood pressure.

North America hormone replacement therapy market is currently being driven by several factors. A surge in the incidences of hormone imbalance disorders, especially in the geriatric and neonatal populations, is spurring the demand for HRT in North America. In line with this, the rising need for new treatment options with better safety results is further catalyzing the market growth in the region.

Apart from this, increasing R&D activities for hormone replacement products is enhancing their quality and efficiency. Additionally, the increasing consumer awareness, coupled with the rising technological innovations, such as new gel-based formulations, have also spurred the demand for hormone replacement products in the region.

Key Questions Answered in This Report:

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Report Insights

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Key Regions Analysed

Market by Product

Market by Route of Administration

Market by Type of Disease

For more information about this report visit https://www.researchandmarkets.com/r/ha9s07

About ResearchAndMarkets.com ResearchAndMarkets.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.

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North America Hormone Replacement Therapy (HRT) Market Research 2024: A $6.9 Billion Industry by 2032, Driven ... - GlobeNewswire

Recommendation and review posted by Bethany Smith

One in 36 children are diagnosed with Autism Spectrum Disorder. But of those, very few actually know what specific gene mutation may be behind their diagnosis. National Health Reporter Erin Billups takes a look at how early genetic testing can improve treatment options and quality of life for patients.

He's happy. He's outgoing. He's more outgoing with adults than he is with children. He loves water play. Loves to swim, said Genie Egerton-Warbuton, who cant help but smile herself when she speaks about her 11-year-old son Rowland.

Like many expecting parents, during her pregnancy with Rowland, Egerton-Warbuton underwent prenatal testing.

I had to have a cesarean section. I'll never forget the doctor saying, you've given birth to a perfect, beautiful baby. Healthy, you know, healthy little boy that we know doesn't have any sort of issues or genetic abnormalities, etc. from this test, said Egerton-Warbuton.

But from his notched eyelids at birth to missed developmental milestones, Egerton-Warbuton, a former preschool teacher, knew something wasnt right.

It's not normal for a child not to walk until they're two. And even his gait wasn't appropriate. And he was able to say some words, but then he was losing the words. He was able to pick up food and kind of feed himself. But then the next day, he needed to be spoon fed. It was just kind of, things did not feel right, said Egerton-Warbuton.

When Rowland was four, he underwent another round of genetic testing and was finally diagnosed with a form of autism called ADNP syndrome a deficiency of the activity-dependent neuroprotective protein a mutation that wasnt included in the test when Rowland was in utero.

Certain characteristics are common in ADNP syndrome. (Spectrum News)

We know that protein is supposed to play a really important role in brain development, said Dr. Alex Kolevzon, director of the Seaver Autism Center at Mount Sinai Hospital in New York City, of the ADNP deficiency. The hallmarks of ADNP syndrome, like autism, are social communication problems.

Kolevzon said ADNP is now included on the genetic screening test for autism, along with more than 200 other known gene mutations that cause varying degrees of autism. ADNP accounts for about 0.2% of autism cases, but there may be even more who have it. Kolevzon says it is time for more families to get their kids tested.

Oftentimes, families don't fully appreciate the value of genetic testing. You know, they have a child, the child that has autism. And it's not clear how knowing what the genetic causes will actually impact the child's life, said Kolevzon.

The sooner families and doctors know, the better, said Kolevzon. While not every case of autism will have a genetic abnormality, its estimated that about 10 to 30 percent will find a known cause through testing.

For us, there's a kind of a shift towards more personalized medicine. And so, you know, if you know exactly what's wrong with the biology, it gives you an opportunity to develop more targeted treatments, said Kolevzon.

Rowland was enrolled in a study led by Kolevzon, with nine other kids with ADNP Syndrome.

Each child was given a very low dose of ketamine. Kolevzon said the drug may promote synaptic plasticity or nerve cell growth. Thats what they eventually found, participants saw improvement in sensory sensibilities, hyperactivity and repetitive behaviors. Larger studies are needed to confirm the findings.

After the infusion, the next day he said, Mommy, which was, you know, amazing, said Egerton-Warbuton. And we noticed that he was able to navigate a city street without walking into people.

Rowland Egerton-Warburton has been working on his communication. (Courtesy Genie Egerton-Warbuton)

Rowland continues to work on further developing his speech. He is also learning to use a device that will help him communicate.

Egerton-Warbuton said her hope is that as more people with autism and other brain-based disorders are tested, more resources will go toward finding transformative genetic therapies for ADNP and other disorders.

So many of these hospitals do want to collaborate and do want to help these children. But it's very hard when you have a small amount of children and adults, said Egerton-Warbuton. You just feel kind of stuck.

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Early genetic testing could help children with autism - Spectrum News NY1

Recommendation and review posted by Bethany Smith

According to latest study, the U.S. precision medicine market size was estimated at USD 24.95 billion in 2023 and is projected to hit around USD 76.12 billion by 2033, growing at a CAGR of 11.80% during the forecast period from 2024 to 2033.

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Advancements in precision medicine have catalyzed significant growth in the U.S. market, leading to groundbreaking discoveries and FDA-approved treatments tailored to individual characteristics such as genetic makeup or tumor profiles. Routine molecular testing in cancer care empowers physicians to select treatments that enhance survival rates and minimize adverse effects for patients. However, the effectiveness of precision care hinges on the quality of diagnostic tests guiding treatment decisions.

The U.S. precision medicine market is experiencing rapid growth fueled by innovative approaches to disease prevention and treatment, such as personalized medicine. This approach considers individual differences in genes, environments, and lifestyles, with the goal of delivering targeted treatments to patients at the right time. The FDA plays a crucial role in ensuring the accuracy of Next Generation Sequencing (NGS) tests, which generate vast amounts of information posing novel regulatory challenges. To address this, the FDA has collaborated with industry stakeholders, laboratories, academia, and patient/professional societies to develop a flexible regulatory framework. This approach leverages consensus standards and state-of-the-art computing technology to support NGS test development, fostering innovation and accelerating access to reliable genetic tests. The Precision Medicine Initiative, led by the NIH, aims to understand how genetics, environment, and lifestyle impact disease prevention and treatment. Short-term goals focus on expanding precision medicine in cancer research, while long-term objectives aim to integrate precision medicine into all areas of healthcare. The All of Research Program, involving at least 1 million volunteers, underscores the initiative's commitment to large-scale precision medicine implementation nationwide.

U.S. Precision Medicine Market Key Takeaways

The Global Precision Medicine Market Size and share 2024 to 2033.

The global precision medicine market size is calculated at USD 91.72 billion for 2024 and is expected to reach around USD 246.30 billion by 2033, growing at a CAGR of 11.6% from 2024 to 2033, The North America market has captured 49.19% of the total revenue share in 2023.

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U.S. Precision Medicine Market Dynamics

Driver

Empowering Healthcare

Precision medicine, synonymous with personalized care, empowers healthcare providers to tailor specific treatments based on individual genetic, protein, and biological profiles. Particularly in cancer care, precision medicine focuses on how genetic or protein alterations in cancer cells influence treatment options. Beyond oncology, precision medicine offers versatile applications, leveraging lab test insights to craft personalized care plans with specific recommendations. This approach not only enhances diagnostic accuracy and treatment efficacy but also facilitates informed decisions regarding lifestyle modifications and preventive measures to mitigate cancer risks. As precision medicine continues to gain traction, its role in optimizing patient care drives growth in the U.S. precision medicine market, offering promising outcomes for patients and fostering innovation in healthcare delivery.

Restraint

Cost Challenges

The substantial costs associated with precision therapy, often reaching thousands or tens of thousands of dollars per month, exacerbate the financial burden of cancer diagnosis. Research indicates that a significant proportion of patients exhaust their assets within two years of diagnosis. The Center for Medicare and Medicaid Services' decision to cover genetic testing for advanced cancer patients is expected to incur an additional annual cost of $2.5 billion for the agency. These financial constraints pose challenges to the growth of the U.S. precision medicine market, hindering accessibility and affordability of innovative treatments for patients.

Opportunity

Advanced technologies for precision medicine

The surge in precision medicine technologies presents a compelling opportunity to enhance targeted care, potentially improving patient outcomes. However, this transformation also introduces complexities for health systems. As these technologies gain traction in routine clinical practice, they have the potential to revolutionize various aspects of care delivery, including care pathways, healthcare infrastructure, and patient experiences. The adoption of precision medicine is likely to raise equity considerations, ensuring equitable access to innovative treatments. Embracing these advancements presents an opportunity for growth in the U.S. precision medicine market, driving innovation and reshaping the healthcare landscape for the better.

Recent trends in U.S. precision medicine market:

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Report Highlights

By Technology

The U.S. precision medicine market is segmented into bioinformatics, big data analytics, drug discovery, gene sequencing, companion diagnostics, and other categories. The drug discovery segment stands out as the dominant force in this market. Precision medicine holds immense promise in enhancing the success rates of Phase II and III clinical trials by tailoring treatment options to patient subgroups based on molecular profiles, lifestyle, and environmental factors. Since its inception, precision medicine has witnessed a rapid expansion across various medical and healthcare applications, with oncology leading the charge in its implementation. This paradigm shift is revolutionizing the drug development process by pinpointing targets responsible for diseases in individual patients and stratifying clinical trials based on underlying mechanistic causes. As precision medicine continues to evolve, it is poised to drive innovation and advancements in the U.S. healthcare landscape, paving the way for more effective and personalized treatment approaches.

Precision medicine revolutionizes clinical trials by stratifying patients based on their genetic and molecular profiles, enabling more targeted and effective treatment approaches. This approach integrates clinical and molecular patient data to decipher the biological underpinnings of diseases, ultimately aiming for optimal patient outcomes. Clinical trials serve as the cornerstone for scientifically evaluating investigational agents, devices, and biologics, ranging from chemotherapy agents to gene therapies, in human volunteers to assess safety and efficacy. By leveraging precision medicine in clinical trial design, researchers can enhance trial efficacy and accelerate the development of innovative therapies tailored to individual patient needs, driving advancements in healthcare and pharmaceutical industries.

By Application Insights

The U.S. precision medicine market is segmented into CNS, immunology, oncology, respiratory, and other categories, with oncology emerging as the dominant sector. Precision medicine, as defined by the US National Cancer Institute, utilizes genetic, protein, and environmental information to prevent, diagnose, and treat diseases. While definitions may vary among stakeholders, targeted drug therapy, precision radiotherapy, and surgery are increasingly prevalent in clinical practice for both solid and hematological cancers. Precision medicine plays a pivotal role in modern cancer care, with comprehensive molecular profiling of tumors essential for identifying targetable abnormalities or biomarkers. Lung cancer exemplifies the significance of precision medicine, with genomic alterations soon expected to guide therapy in the majority of cases. As precision medicine continues to advance, its application in oncology promises to revolutionize cancer treatment, improving patient outcomes and driving growth in the U.S. precision medicine market.

By End-Use

In the U.S. precision medicine market, segmentation by end-users includes diagnostic companies, pharmaceutical companies, healthcare IT companies, and others, with pharmaceutical companies emerging as the dominant segment. Precision medicine is poised to revolutionize the entire pharmaceutical value chain, influencing early development stages through to go-to-market strategies. The next five years represent a critical window for pharmaceutical companies to capitalize on this transformative potential, necessitating proactive engagement and risk-taking across the healthcare ecosystem. Beyond enhancing disease detection, diagnosis, and treatment, precision medicine holds the promise of preventive healthcare by leveraging analytics to identify patient risks before they manifest. This proactive approach not only improves patient outcomes but also has the potential to lower costs for healthcare systems, underscoring the imperative for pharmaceutical companies to embrace precision medicine and drive innovation in the U.S. market.

By Sequencing Technology

In the U.S. precision medicine market, segmentation by sequencing technologies includes pyrosequencing, sequencing by synthesis, sequencing by ligation, single-molecule real-time sequencing (SMRT), ion semiconductor sequencing, chain termination sequencing, and nanopore sequencing, with the SMRT segment poised to dominate over the forecast period. While short-read massive parallel sequencing has become a standard diagnostic tool in medicine, it faces inherent limitations such as GC bias and difficulties in mapping to repetitive elements and discriminating analogous sequences. Single molecule real-time sequencers address these challenges by offering long-read capabilities, resulting in higher consensus accuracies and improved detection of epigenetic modifications from native DNA. As precision medicine continues to evolve, the adoption of SMRT sequencing technologies promises to enhance the accuracy and reliability of genetic analyses, driving advancements in personalized healthcare and shaping the future of genomic medicine in the U.S. market.

By Product

In the U.S. precision medicine market, segmentation into consumables, instruments, and services reveals consumables as the leading segment. Precision medicine strategies revolutionize healthcare by delving deeply into patients' genetic and genomic data, enabling accurate disease prediction and effective prevention, diagnosis, and treatment. This approach empowers physicians to select sensitive drugs, optimal dosages, and timing for medication usage, while minimizing adverse side effects. Consumables play a pivotal role in facilitating precision medicine implementation, providing the essential tools and materials required for genetic testing, sequencing, and analysis. As precision medicine continues to gain momentum, the consumables segment is poised to drive significant growth, enabling healthcare providers to deliver personalized and targeted therapies to patients, ultimately improving patient outcomes and revolutionizing the healthcare landscape.

By Route of Administration

In the U.S. precision medicine market, segmentation by route of administration reveals oral medication as the leading segment. Oral administration offers convenience, cost-effectiveness, and widespread acceptance among patients, making it the most commonly used medication administration route. Typically, the small intestine serves as the primary site of drug absorption, with medication bioavailability influenced by the rate and extent of absorption across the intestinal epithelium. This route of administration is particularly suitable for patients capable of ingesting and tolerating oral medication. Additionally, some medications with short half-lives are formulated as timed-release or sustained-release forms, allowing for gradual absorption over several hours. As precision medicine continues to advance, oral medication remains a cornerstone in delivering targeted therapies to patients, driving growth and innovation in the U.S. market.

By Drugs Insights

In the U.S. precision medicine market, segmentation by drug category includes Alectinib, Osimertinib, Mepolizumab, Aripiprazole Lauroxil, and others, with the Mepolizumab segment anticipated to experience significant growth. Mepolizumab, an anti-IL-5 monoclonal antibody developed for severe eosinophilic asthma treatment, exemplifies a clinical development program shaped by robust scientific principles. Initially, clinical data on mepolizumab's impact on lung function in a general asthmatic population were underwhelming. However, subsequent research revealed its effectiveness in reducing asthma exacerbations, particularly in patients with severe disease. Advancements in understanding asthma pathobiology further identified a target population and predictive biomarkers for mepolizumab. As precision medicine continues to evolve, mepolizumab's tailored approach to treating severe eosinophilic asthma positions it for substantial growth, offering promising outcomes for patients and driving innovation in the U.S. market.

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U.S. Precision Medicine Market Recent Developments

Related Report

U.S. Precision Medicine Market Top Key Companies:

U.S. Precision Medicine Market Report Segmentation

This report forecasts revenue growth at country levels and provides an analysis of the latest industry trends in each of the sub-segments from 2021 to 2033. For this study, Nova one advisor, Inc. has segmented the U.S. Precision Medicine market.

By Technology

By Application

By End-Use

By Sequencing Technology

By Product

By Route of Administration

By Drugs

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U.S. Precision Medicine Market Size to Hit USD 76.12 Billion by 2033 - BioSpace

Recommendation and review posted by Bethany Smith

Only have a minute? Listen instead

Former U.S. Rep. Mayra Flores wants mandatory DNA testing of all immigrants and favors a return of the much-denounced policy of separating children from their parents at the border.

The Donald Trump administration utilized both practices and likely would resume them if he is elected in November; Trump has said he plans to resurrect all of his previous immigration policies and impose new ones that would be more severe, including the largest domestic deportation operation in Americas history.

Flores, who is running to regain the congressional seat she lost in 2022, defends those policies, although family separation has roundly been denounced as inhumane. Some of the children were mere infants. Worse, many of those children have not been reunited with their parents half a decade later.

Until recently, our stated immigration policy was to keep families together. Federal law prohibits keeping immigrant children in detention for more than 20 days. The Trump administration bypassed that law by reclassifying separated children as unaccompanied minors in order to detain them indefinitely.

Obviously the law was violated in spirit and it doesnt seem to matter to Flores, who seeks to return to Congress to enact such laws. In fact, Republican lawmakers have filed legislation that would mandate genetic testing of immigrants. Our own Sen. Ted Cruz submitted the bill in that chamber.

I dont care if these children are with us for months, she said at a February gathering of the conservative youth group Turning Point USA in Brownsville.

Flores said longer child detentions might be necessary to allow time for the DNA testing, which she says is needed to verify that the children in fact are related to the people who brought them.

Genetic testing shouldnt be needed for such evaluations; it shouldnt be hard to see a difference in a childs comportment with a parent or guardian as opposed to a total stranger.

Its also obvious that any DNA testing that was done in the past didnt work. Thousands of families remain separated and might never be reunited; records of those tests and related detentions if kept at all were so shoddy that officials continue looking for detained childrens parents and cant find them.

Moreover, laboratories across the country that would perform such tests would have difficulty handling the burden not to mention the expense. Labs currently are so backlogged with forensic testing that criminal cases are being delayed while they wait for evidence.

Widespread testing during the Trump term was slowed down further because the demand for the tests outpaced the supply. Trump and his supporters constantly throw out the term family values in their campaigns. Shredding families, regardless of their nationality or legal status, obviously clashes.

Keeping migrant families together isnt just the right thing to do, it seems the most practical and efficient. Processing them together should speed up the process and ensure that they receive visas together if they qualify, or are deported together if they dont.

Republicans penchant for punitive measures increasingly is defying not only practicality, but morality.

Continued here:
Editorial: DNA testing of immigrants more trouble than it's worth, shouldn't even be necessary - MyRGV

Recommendation and review posted by Bethany Smith

There is a lot of beauty to aging as well as some complications, with Alzheimer's disease being a complicated condition that affects millions worldwide and older generations in particular. With about five million Americans grappling with Alzheimer's disease, its prevalence appears to be steadily climbing. It is projected to soar to eight figures in the future, potentially impacting over 13 million people by 2050. This taxing disease takes a toll on those who get it and their families, which has researchers and everyday people eager to find solutions. Without an existing cure for Alzheimer's disease, preventative measures have become the focus.

With questions arising surrounding the disease's genetic implications, people can turn to Alzheimer's genetic testing, although there is debate surrounding the pros and cons. Planning for the future, contributing to research efforts, mitigating personal risk factors, and finding solace in negative results are among the arguments in favor of testing. However, a cautious approach is also necessary, considering the multifaceted implications of genetic revelations and their potential to be inaccurate in determining if Alzheimer's is in one's future.

Genetics undeniably shapes Alzheimer's risk, yet the clarity genetic testing offers remains elusive for many. The apolipoprotein E gene, particularly the APOE e4 variant, is a significant genetic risk factor. Still, its presence doesn't guarantee Alzheimer's onset, complicating counseling for those tested. Moreover, while rare genetic mutations directly cause Alzheimer's, they're predominantly observed in familial early-onset cases, leaving more ambiguity for the broader population. Lifestyle factors also intertwine with genetic predispositions, which influence Alzheimer's susceptibility. Conditions like obesity, hypertension, and diabetes, coupled with specific gene mutations, elevate the risk across varied demographics.

While other medical conditions have made substantial strides in discovering cures, breakthroughs in Alzheimer's treatment have not been as evident. For over a decade, there's been an absence of new drugs that bring promising results, shedding light on the challenges researchers face in curing Alzheimer's once and for all. More positive developments include the recent progress in using brain and spinal fluid proteins to predict Alzheimer's progression. This advancement offers some promise for future intervention.

Despite these advancements, an added dose of anxiety often accompanies genetic testing, given its uncertainty. Alzheimer's disease is not a fast-acting condition, as the progressive form of dementia is known to manifest gradually. Even with impaired memory, cognition, and behavior, a definitive diagnosis is still only cemented during postmortem brain examinations, despite visible symptoms while the afflicted is alive. When symptoms of Alzheimer's disease start to become evident, varied tests aid in assessing cognitive functionand ruling out alternative diagnoses to guide treatment decisions.

Alzheimer's disease includes several stages, and navigating each stage of the disease demands comprehensive caregiving strategies that acknowledge the diverse challenges each phase presents. While a cure is not yet available, many resources exist to equip families and caretakers to offer tailored support and quality of life to those with Alzheimer's disease. The daunting possibility of Alzheimer's disease, from the mild cognitive impairment stage to the final stage of advanced dementia, can draw people to seek answers through genetic testing. Researchers continue to seek answers as lifestyle modifications and healthy living habits currently stand as effective ways to combat cognitive decline.

Disclaimer: The San Francisco Weekly newsroom and editorial were not involved in the creation of this content.

Link:
Genetic Testing for Alzheimer's: Navigating Risks and Prevention Strategies - SF Weekly

Recommendation and review posted by Bethany Smith

MELBOURNE, Australia, May 29, 2024 (GLOBE NEWSWIRE) -- Genetic Technologies Limited (ASX: GTG; NASDAQ: GENE, Company, GTG), a global leader in genomics-based tests in health, wellness and serious disease,

is pleased to announce the Company has released its novel Comprehensive Risk Assessment test that covers 100% of women at risk of developing breast and ovarian cancer. The innovative test, available to all women above the age of 30, assesses a womans risk of cancer due to hereditary, including those with common gene mutations, and sporadic disease.

Launched at the inaugural Know Your Risk event in California, USA, the event provided invaluable insights of the role of genomics in womens health. Co-hosted by Dr Kristi Funk, known for her surgical treatment of celebrity Angelina Jolie, alongside CEO & Founder of Humanise Health, Krystal Barter, the event also featured panellists such as US based OB-GYN Dr. Carolynn Young, Andrea Hans, Allyn Rose Oertel, and Matthew Zachary.

This new geneType Comprehensive Risk Assessment test represents a significant leap in preventative healthcare, whereby clinicians will have a complete understanding of their patients risk profile of developing one of the deadly cancers. The addition of the germline component to GTGs platform provided the ability to screen 100% of women at risk. Retrospective data shows 5% to 10% of breast and ovarian cancers are caused by gene mutations and the remainder are due to sporadic condition.

This represents a massive market opportunity for geneType, with 1 in 8 chances of a woman in the US developing breast cancer equating to about 310,000 diagnosed cases annually. Although less prevalent, ovarian cancer accounts for 19,600 cases diagnosed yearly in the US and claims 12,700 deaths annually. Ovarian cancer is more deadly and is among the leading cause of cancer deaths in women. Dr Kirsti Funk noted that Understanding your cancer risk based on critical diet and lifestyle and genetic factors allows me to create a more directed, effective risk-reducing strategy.

This advancement in personalised preventative healthcare, was welcomed by the companys strategic partners in employer groups, functional medicine clinics and existing physician networks. The OB-GYNs are overwhelmingly excited about this launch and expressed their satisfactions on the abilities of our new comprehensive test for early diagnosis and ultimately saving lives.

GTGs CEO, Simon Morriss, said We are incredibly proud to have been part of this Know Your Risk event which underscored the importance of genetic testing and risk assessment in transforming womens health, inspiring attendees to advocate for their health and well-being proactively. We are also very fortunate and to have the opportunity to launch this unique risk assessment test in the presence of such a wonderful group of people.

Peter Rubinstein, GTGs Chairman commented, We are also excited to announce that in preparation for significant demand for this revolutionary test we have fully onboarded a high throughput automated laboratory in the United States with capacity to scale up to run 100,000 tests per month should future demand require. Every woman deserves the right to know her risk.

For more information, please visit http://www.genetype.com.

Authorised for release by the Board of Directors.

Enquiries Simon Morriss Chief Executive Officer E: investors@genetype.com

About Genetic Technologies Limited

Genetic Technologies Limited (ASX: GTG; Nasdaq: GENE) is a diversified molecular diagnostics company. A global leader in genomics-based tests in health, wellness and serious disease through its geneType and EasyDNA brands. GTG offers cancer predictive testing and assessment tools to help physicians to improve health outcomes for people around the world. The company has a proprietary risk stratification platform that has been developed over the past decade and integrates clinical and genetic risk to deliver actionable outcomes to physicians and individuals. Leading the world in risk prediction in oncology, cardiovascular and metabolic diseases, Genetic Technologies continues to develop risk assessment products. For more information, please visit http://www.genetype.com

Forward-Looking Statements

This announcement may contain forward-looking statements about the Companys expectations, beliefs or intentions regarding, among other things, statements regarding the expected use of proceeds. In addition, from time to time, the Company or its representatives have made or may make forward-looking statements, orally or in writing. Forward-looking statements can be identified by the use of forward-looking words such as believe, expect, intend, plan, may, should or anticipate or their negatives or other variations of these words or other comparable words or by the fact that these statements do not relate strictly to historical or current matters. These forward-looking statements may be included in, but are not limited to, various filings made by the Company with the U.S. Securities and Exchange Commission, press releases or oral statements made by or with the approval of one of the Companys authorized executive officers. Forward-looking statements relate to anticipated or expected events, activities, trends or results as of the date they are made. As forward-looking statements relate to matters that have not yet occurred, these statements are inherently subject to risks and uncertainties that could cause the Companys actual results to differ materially from any future results expressed or implied by the forward-looking statements. Many factors could cause the Companys actual activities or results to differ materially from the activities and results anticipated in such forward-looking statements as detailed in the Companys filings with the Securities and Exchange Commission and in its periodic filings with the ASX in Australia and the risks and risk factors included therein. In addition, the Company operates in an industry sector where securities values are highly volatile and may be influenced by economic and other factors beyond its control. The Company does not undertake any obligation to publicly update these forward-looking statements, whether as a result of new information, future events or otherwise, except as required by law.

_____________________________ https://www.cancer.org/cancer/types/ovarian-cancer/about/key-statistics.html https://www.instagram.com/p/C7XX-sKygdG/?utm_source=ig_web_copy_link

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GTG Launches Revolutionary geneType Test covering 100% Risk of Breast and Ovarian Cancer - GlobeNewswire

Recommendation and review posted by Bethany Smith

image:

Photograph of a stick insect.

Credit: Filippo Castellucci.

Traits are often lost during evolution, either because they are no longer beneficial or because they are too costly to maintain. When this happens, it is generally believed that the genes underlying the trait will eventually degrade as well, making it difficult if not impossible for the trait to re-emerge. Yet, there are numerous examples in nature of once-lost traits reappearing in descendent lineages. According to Giobbe Forni, a Research Fellow at the University of Bologna, Mapping the presence and absence of traits onto a species tree suggests that some traits may have been lost in the lineages leading to extant species and then subsequently reinstated. Wings in stick insects are considered one of the more iconic instances of this evolutionary process. This implies that the genes underlying these traits may be preserved, in some cases for millions of years. Unfortunately, research on the molecular basis of such re-emergence is sparse, leaving the underlying mechanisms responsible for such preservation largely open to speculation until now. In anew study published in Genome Biology and Evolution, Forni and his colleagues shed light on another complex trait that has been lost in some stick insectsthe production of males. Loss of the ability to produce males results in populations of only females, which reproduce by parthenogenesis, a form of asexual reproduction. The study reveals that genes that are highly connected in regulatory networks and involved in multiple biological processes may be maintained long after a trait is lost, providing a potential avenue for trait re-emergence over long evolutionary time scales.

In the new study, Forni and his co-authors Barbara Mantovani, Alexander S. Mikheyev, and Andrea Luchetti performed a comparative analysis of three species of stick insects in the genusBacillus. WhileBacillus grandii marettimipopulations are composed of males and females that reproduce sexually,Bacillus atticuspopulations have lost the ability to produce males, comprising only females that reproduce by parthenogenesis. A third species,Bacillus rossius, includes both sexual populations and parthenogenetic populations that have lost the ability to produce males. By studying the fates of genes involved in male reproduction in these three species, the authors sought to investigate the extent to which genes are preserved after trait loss and the potential mechanisms driving this preservation.

The researchers first identified gene networks whose expression was correlated with either male or female reproduction in the sexual speciesB. marettimiand then evaluated the same genes inB. atticusandB. rossius. Surprisingly, male-related genes exhibited no signs of weakened selection or accelerated evolution compared with female-related genes in the parthenogenetic species. Furthermore, male-related patterns of gene expression were partially preserved across both parthenogenetic species.

Delving deeper, the researchers found that genes in female-related networks were primarily expressed in female reproductive tissues, while those in male-related networks were expressed in maleandfemale reproductive tissues, including both sexual and parthenogenetic females. This suggests that male-related genes may also play roles in female reproduction. The involvement of a gene in multiple biological processes is known as pleiotropy, and this phenomenon may explain the preservation of male-related genes in these parthenogenetic stick insects, as previously hypothesized.

Moreover, the authors found that genes that were highly connected to many other genes in the network were more likely to be expressed in the reproductive tissues of parthenogens, suggesting that a gene's network connectivity may also influence its gene preservation after trait loss. Taken together, these findings indicate that the molecular ground plan of the once-lost male reproductive process may persist due to pleiotropic effects on other traits, explains Forni. Different genes may undertake different trajectories of preservation and decay depending on the level of pleiotropy within the gene regulatory network.

This study not only sheds light on genetic architecture persistence after trait loss but also offers a potential glimpse into the emergence of rare males and cryptic sex (i.e. episodic generation of males and sexual reproduction), which have been observed in an increasing number of lineages that were thought to have lost the ability to produce males long ago. This opens up new potential avenues for research, with implications that may reach far beyond stick insects. Looking at how widespread genetic preservation after trait loss is on a larger scale remains fundamental. Although theBacillusspecies complex offers a nice framework to address these issues, it would be useful to analyze a larger species complex where multiple transitions between reproductive strategies has occurred, notes Forni. While it is often necessary to rely on model species to discover and dissect biological processes, it is even more important to test our hypotheses in a wider context. This will be possible only if we dedicate more effort to observing and analyzing the amazing diversity of organisms and their intricate adaptations.

Genome Biology and Evolution

Observational study

Animals

Parthenogenetic stick insects exhibit signatures of preservation in the molecular architecture of male reproduction

21-May-2024

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

Excerpt from:
Maintenance of male-related genes after loss of males in stick insects - EurekAlert

Recommendation and review posted by Bethany Smith

Fortum, a Nordic energy company with headquarters in Espoo, Finland, near Helsinki, and offices in 10 other countries, announced that it will modernize the Loviisa nuclear power plants low-pressure turbines. The project will start in 2026 as part of lifetime extension-related investments at the site.

The Lovissa plant is a dual-unit facility with a total net capacity of about 1,014 MW (Figure 1). Unit 1 was commissioned in 1977 and Unit 2 in 1980. The plant is located about 100 kilometers east of Helsinki and provided about 10% of Finlands electricity production in 2023, with the units operating at capacity factors of 89.79% and 92.32%, respectively. The modernization of the turbines is expected to increase the total capacity of the plant by approximately 38 MW.

In February 2023, the Finnish government granted a new operating license for the power plant until 2050. As a result, Fortum expects to perform continuous improvements to ensure reliable electricity production for at least the next 26 years. Fortum said it has invested approximately 200 million in refurbishing the Loviisa power plant over the past five years, and it estimated that investments related to the lifetime extension will amount to about 1 billion by 2050.

Extending the lifetime of the power plant is a major investment with a positive impactboth economically and in terms of employment. Modernizing the low-pressure turbines is our first significant investment in preparation for the lifetime extension. Our aim is for the power plant to operate during the new operating license period just as stably, reliably, and safely as it has so far, Sasu Valkamo, senior vice president of the Loviisa power plant, said in a statement.

Doosan koda Power was selected as the supplier for the low-pressure turbine job. The modernization will be carried out in conjunction with normal annual outages. In the turbine project, eight low-pressure turbine housings and their internal parts will be renewed. This particular modernization is targeting only the turbines, so it wont impact the reactor plant or nuclear safety. The project is expected to significantly improve the efficiency of the turbine plants electricity production without increasing the thermal output of the reactor.

Doosan koda Power is a seasoned turbine supplier, and we have good experiences working with them. In our previous modernization project, Doosan koda Power also supplied us with high-pressure turbines, Valkamo said.

Radek Trnn, head of Sales Nuclear at Doosan koda Power, said, Fortum is our long-term customer and we are very proud to be part of this new important modernization project at the Loviisa nuclear power plant. Nuclear power is a strategic segment for us, and this contract is further confirmation that we are on the right track.

Doosan koda Power is a leading global manufacturer and supplier of power plant machinery, especially steam turbine-generator sets with outputs between 3 MW and 1,200 MW. The company is part of the Doosan Group, which supplies technologies and services to customers all over the world.

Fortum, meanwhile, has more than 150 power plants in its fleet. In addition to the Lovissa plant, Fortum also owns shares in Olkiluoto Units 1, 2, and 3, Oskarshamn Unit 3, and Forsmark Units 1, 2, and 3. Fortum also has hydro, combined heat and power (CHP), solar, and wind power plants. It touts a 98% CO2-free electricity generation portfolio. It is the third-largest power generator in the Nordic countries and one of the leading heat producers globally.

Aaron Larson is POWERs executive editor (@POWERmagazine).

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Patients characteristics

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Structural dataset and computational analysis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

T

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

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

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

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

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

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

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

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

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

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

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Plasmid design and construction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Summary of library contents are in Supplementary Fig. 18.

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

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

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

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

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

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

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

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

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

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

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

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

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Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations - Nature.com

Recommendation and review posted by Bethany Smith

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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CRISPR Therapeutics Strengthens Executive Leadership Team with Key Appointments - GlobeNewswire

Recommendation and review posted by Bethany Smith

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

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

Check Out Our Latest Report on CRSP

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

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

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

(Get Free Report)

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

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

Recommendation and review posted by Bethany Smith

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Recommendation and review posted by Bethany Smith

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

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

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

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

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

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

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

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

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

Recommendation and review posted by Bethany Smith

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Recommendation and review posted by Bethany Smith

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Recommendation and review posted by Bethany Smith