Gene Therapy For Sickle Cell Disease Research Articles

Revolutionary breakthrough: FDA approves CASGEVY™, the first CRISPR/Cas9 gene therapy for sickle cell disease

Revolutionary breakthrough: FDA approves CASGEVY™, the first CRISPR/Cas9 gene therapy for sickle cell disease

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Current state of gene therapy in sickle cell disease.

Sickle cell disease (SCD) is a type of hemoglobinopathy due to an autosomal recessive genetic defect, causing significant red cell sickling, multi-organ damage and long-term severe morbidities. Due to its complicated care and the impact on quality of life, a curative treatment for SCD is highly desirable. In recent years, gene therapy is emerging as a curative option for SCD, where autologous haematopoietic stem cells are collected from SCD patients and genetically modified ex vivo to reduce its sickling tendency before reinfusion. Although still largely investigational, a limited number of gene therapy options have been recently granted approval for SCD patients. Published data are still currently limited, but early studies have so far demonstrated the intended outcomes of less vaso-occlusive crisis and haemolysis. Nonetheless, despite its curative potential, larger clinical trials and longer follow-up period are still necessary to evaluate the safety of this treatment option, especially the risk of unintended genetic modifications. Furthermore, SCD patients frequently have limited access to specialty care; hence, the issues of affordability and accessibility to SCD gene therapy must also be addressed for it to benefit the appropriate patient population.

The value-based price of transformative gene therapy for sickle cell disease: a modeling analysis

Sickle cell disease (SCD) is an inherited, progressively debilitating blood disorder. Emerging gene therapies (GTx) may lead to a complete remission, the benefits of such can only be realized if GTx is affordable and accessible in the low-and middle-income countries (LMIC) with the greatest SCD burden. To estimate the health impacts and country-specific value-based prices (VBP) of a future gene therapy for SCD using a cost-utility model framework. We developed a lifetime Markov model to compare the costs and health outcomes of GTx versus standard of care for SCD. We modeled populations in seven LMICs and six high-income countries (HICs) estimating lifetime costs and disability-adjusted life-years (DALYs) in comparison to estimates of a country’s cost-effectiveness threshold. Each country’s unique VBP for GTx was calculated via threshold analysis. Relative to SOC treatment alone, we found that hypothetical GTx reduced the number of people symptomatic with SCD over time leading to fewer DALYs. Across countries, VBPs ranged from $3.6 million (US) to $700 (Uganda). Our results indicate a wide range of GTx prices are required if it is to be made widely available and may inform burden and affordability for ‘target product profiles’ of GTx in SCD.

Gene Therapy Versus Common Care for Eligible Individuals With Sickle Cell Disease in the United States : A Cost-Effectiveness Analysis.

Sickle cell disease (SCD) and its complications contribute to high rates of morbidity and early mortality and high cost in the United States and African heritage community. To evaluate the cost-effectiveness of gene therapy for SCD and its value-based prices (VBPs). Comparative modeling analysis across 2 independently developed simulation models (University of Washington Model for Economic Analysis of Sickle Cell Cure [UW-MEASURE] and Fred Hutchinson Institute Sickle Cell Disease Outcomes Research and Economics Model [FH-HISCORE]) using the same databases. Centers for Medicare & Medicaid Services claims data, 2008 to 2016; published literature. Persons eligible for gene therapy. Lifetime. U.S. health care sector and societal. Gene therapy versus common care. Incremental cost-effectiveness ratios (ICERs), equity-informed VBPs, and price acceptability curves. At an assumed $2 million price for gene therapy, UW-MEASURE and FH-HISCORE estimated ICERs of $193 000 per QALY and $427 000 per QALY, respectively, under the health care sector perspective. Corresponding estimates from the societal perspective were $126 000 per QALY and $281 000 per QALY. The difference in results between models stemmed primarily from considering a slightly different target population and incorporating the quality-of-life (QOL) effects of splenic sequestration, priapism, and acute chest syndrome in the UW model. From a societal perspective, acceptable (>90% confidence) VBPs ranged from $1 million to $2.5 million depending on the use of alternative effective metrics or equity-informed threshold values. Results were sensitive to the costs of myeloablative conditioning before gene therapy, effect on caregiver QOL, and effect of gene therapy on long-term survival. The short-term effects of gene therapy on vaso-occlusive events were extrapolated from 1 study. Gene therapy for SCD below a $2 million price tag is likely to be cost-effective when applying a societal perspective at an equity-informed threshold for cost-effectiveness analysis. National Heart, Lung, and Blood Institute.

Gene therapy for sickle cell disease.

Sickle cell disease (SCD) is potentially curable after allogeneic hematopoietic stem cell transplantation (HSCT) or autologous HSCT after ex vivo genetic modification. Autologous HSCT with gene therapy has the potential to overcome many of the limitations of allogeneic HSCT that include the lack of suitable donors, graft-versus-host disease, the need for immune suppression, and the potential for graft rejection. Significant progress in gene therapy for SCD has been made over the past several decades, now with a growing number of clinical trials investigating various gene addition and gene editing strategies. Available results from a small number of patients, some with relatively short follow-up, are promising as a potentially curative strategy, with current efforts focused on continuing to improve the efficacy, durability, and safety of gene therapies for the cure of SCD.

Assessing Psychosocial Readiness for Gene Therapy in Sickle Cell Disease: A Consensus Statement

Introduction: Scientific evidence regarding the efficacy and safety of gene therapy for sickle cell disease (SCD) is growing and clinical trials are increasingly available to patients. As a result, clinical trial teams are now challenged with considering the ways in which psychosocial factors may affect patient decisions about pursuing gene therapy and how pre-gene therapy psychosocial functioning may influence long-term patient outcomes. Although data specific to gene therapy for SCD are currently limited, research in other chronic illness populations undergoing major medical procedures (e.g., solid organ and stem cell transplants) suggest that premorbid psychosocial functioning affects decision-making and post-procedure outcomes. Clinical observations support the relevance of considering psychosocial readiness for curative therapies in patients with SCD but there is a lack of guidance regarding how to carry out such evaluations. Therefore, the NHLBI's Cure Sickle Cell Initiative (CureSCi) formed the Patient Readiness and Resilience Working Group to develop consensus recommendations regarding the assessment of psychosocial readiness in the context of gene therapy for SCD. Methods: The NHLBI CureSCi Patient Readiness and Resilience Working Group was formed in September 2020 and consisted of 10 members including mental health clinicians, researchers, and patients and caregivers. The working group met from 2020-2022 to develop best practice recommendations for the assessment of patient readiness and resilience prior to gene therapy for SCD. Recommendations were developed based on available scientific evidence, expert clinical guidance, review of strategic planning documents, and consultation with CureSCi subcommittees and working groups representative of patient, advocate, clinician, and researcher perspectives. A draft of the consensus recommendations was released for public comment in 2022 and final revisions were completed in early 2023. Results: The CureSCi Patient Readiness and Resilience Working Group reached consensus regarding recommended practices for conducting pre-gene therapy assessments of patient readiness in SCD. The working group developed a recommended workflow for assessing patient readiness, including an institutional self-assessment of readiness to support patients' mental health and address psychosocial barriers to care, brief screening of cognitive functioning and health literacy, patient education about clinical trials and gene therapy, assessment of patient readiness and resilience followed by interventions to address areas of concern, and informed consent. Detailed guidance was developed across four primary domains: 1) Effective education delivery; 2) Evaluation of knowledge and understanding; 3) Evaluation of interest and motivation; and 4) Evaluation of psychosocial risk and resilience factors. The working group created a guide to support implementation of pre-gene therapy assessments, which outlines evidence-based strategies for effective patient education, recommended assessment tools, and sample clinical interview questions. Conclusions: As gene therapy for SCD becomes increasingly available to patients, there is a growing need to be prepared to consider readiness and resilience factors that could affect patient well-being before, during, and after treatment. This CureSCi working group developed a set of consensus recommendations regarding the assessment of psychosocial factors in the context of gene therapy for SCD. A thorough guide was developed to aid in implementation of pre-gene therapy assessments across a range of institutional settings. The presentation will review consensus recommendations, describe practical strategies for conducting assessments, and discuss areas for future research.

Looking ahead: ethical and social challenges of somatic gene therapy for sickle cell disease in Africa.

Somatic gene therapy will be one of the most exciting practices of genetic medicine in Africa and is primed to offer a "new life" for persons living with sickle cell disease (SCD). Recently, successful gene therapy trials for SCD in the USA have sparked a ray of hope within the SCD community in Africa. However, the high cost, estimated to exceed 1.5 million USD, continues to be a major concern for many stakeholders. While affordability is a key global health equity consideration, it is equally important to reflect on other ethical, legal and social issues (ELSIs) that may impact the responsible implementation of gene therapy for SCD in Africa. These include informed consent comprehension, risk of therapeutic misestimation and optimistic bias; priorities for SCD therapy trials; dearth of ethical and regulatory oversight for gene therapy in many African countries; identifying a favourable risk-benefit ratio; criteria for the selection of trial participants; decisional conflict in consent; standards of care; bounded justice; and genetic tourism. Given these ELSIs, we suggest that researchers, pharma, funders, global health agencies, ethics committees, science councils and SCD patient support/advocacy groups shouldwork together to co-develop: (1) patient-centric governance for gene therapy in Africa, (2) public engagement and education materials, and (3) decision making toolkits for trial participants. It is also critical to establish harmonised ethicaland regulatory frameworks for gene therapy in Africa, and for global health agencies to accelerate access to basic care for SCD in Africa, while simultaneously strengthening capacity for gene therapy.

Advancements and Challenges in Gene Therapy Approaches for Sickle Cell Disease: A Comprehensive Review

Sickle cell disease (SCD) is an autosomal recessive genetic blood disorder that occurs when both alleles of the HBB gene have mutations, leading to the production of abnormal haemoglobin (HbS). The presence of HbS causes red blood cells (RBCs) to take on the distinctive sickle-shaped form associated with the disease. This, in turn, leads to blockages in blood vessels, decreased blood circulation, and organs’ damage. Traditional treatments such as blood transfusions and hydroxyurea offer relief but come with their own limitations and associated risks. Gene therapy has emerged as a promising paradigm shift in the quest to cure SCD, offering personalised solutions by targeting the genetic root of the disease. This review article explores the principles and recent advancements in gene therapy for SCD. However, before gene therapy can become the main curative strategy for this disease, several challenges need to be overcome including the need for long-term safety and efficacy evaluations. Ongoing research and innovation hold the promise of enhanced treatments and the potential for a widely available gene therapy, ultimately improving the quality of life for individuals living with SCD.

Sickle Cell Disease: From Genetics to Curative Approaches.

Sickle cell disease (SCD) is a monogenic blood disease caused by a point mutation in the gene coding for β-globin. The abnormal hemoglobin [sickle hemoglobin (HbS)] polymerizes under low-oxygen conditions and causes red blood cells to sickle. The clinical presentation varies from very severe (with acute pain, chronic pain, and early mortality) to normal (few complications and a normal life span). The variability of SCD might be due (in part) to various genetic modulators. First, we review the main genetic factors, polymorphisms, and modifier genes that influence the expression of globin or otherwise modulate the severity of SCD. Considering SCD as a complex, multifactorial disorder is important for the development of appropriate pharmacological and genetic treatments. Second, we review the characteristics, advantages, and disadvantages of the latest advances in gene therapy for SCD, from lentiviral-vector-based approaches to gene-editing strategies.

Long-term health outcomes following curative therapies for sickle cell disease.

Treatment options for patients with sickle cell disease (SCD) continue to rapidly expand and evolve. The goal of therapies such as an allogeneic hematopoietic stem cell transplant (HSCT), gene therapy, and gene editing is to cure rather than control SCD. The benefits of these therapies must be accompanied by minimizing long-term adverse health outcomes from SCD and its treatment. SCD can have adverse effects on a variety of organ systems, including the heart, lung, kidney, and reproductive system, leading to high disease burden, morbidity, and premature mortality in both pediatric and adult patients. While curative therapies are being increasingly used, there remains a paucity of data on the long-term health outcomes associated with these treatments in children and adults with SCD. There are data available regarding the effects of HSCT performed largely for malignant diseases, from which data on SCD outcomes may be extrapolated. However, given the significant differences between these 2 populations of patients who undergo HSCT, such extrapolation is imprecise at best. Furthermore, there are currently no published data on long-term health outcomes following gene therapy for SCD due to current short follow-up times. We summarize the limited data reported on health outcomes following HSCT for SCD and emphasize the need for more research within this area.

Democratizing Knowledge for Sickle Cell Disease Gene Therapy: A Community Based Model for Creating Patient Education Materials

Background: While the advancement of genetic therapy for hematological disorders provides tremendous therapeutic promise, it is not without risk. Sickle cell disease (SCD) is at the forefront of the development of novel, genetic therapies. The process of engaging individuals living with SCD to participate in gene therapy clinical trials requires building trust, respect, and partnership with the community, given the history of neglect towards this disease. With the growth of sickle cell disease gene therapy clinical trials and potential of FDA approval for gene therapy treatments, there is a need for understandable, accessible, and actionable patient education materials (PEMs) that are not sponsored by a specific clinical trial. An objective of the Democratizing Knowledge for Sickle Cell Disease Gene Therapy Project was to examine the process of engaging a diverse group of stakeholders representing different interests within the SCD community to develop open-source patient education materials for sickle cell disease gene therapy. Methods: For this project, we used a community-based dialogue model which incorporates participatory democracy with community based public health engagement approaches (Bonham et al PMID: 19451407). This approach was used to engage a multi-disciplinary group of patients, representatives of SCD advocacy groups, hematologists, gene therapy researchers, and industry and government stakeholders to purposefully work in teams over four workshops to create PEMs. Throughout this process, we recorded their deliberations, and collected survey data at three distinct stages of engagement. Results: We dichotomized the data on experiences and engagement levels into two categories: the Research Community, which includes individuals representing industry, research, government, healthcare, academia, and the Patient and Advocacy Community, which includes individuals living with SCD, parents of children with SCD, and individuals representing community-based SCD advocacy groups. Analysis of this longitudinal data from the three distinct points in time identified patterns across the stakeholder groups represented in these workshops. Of the participants in the research community, 68.3% attended all four workshops, while of those in the patient and advocacy community, 90.9% attended all four workshops (Table 1). In the research community, 95.1% expressed feeling satisfied with their participation in these workshops, and similarly, in the patient and advocacy community, 93.0% expressed feeling satisfied with their participation (Table 2). However, when surveyed about views on the role of power dynamics in this process, there were differing sentiments between these two groups. Amongst the research community, the majority felt power dynamics did not influence the workshops (59.3%). In contrast, amongst the patient and advocacy community, the majority felt power dynamics did influence the workshops (55.2%) (Table 2). In addition to this survey data, further data regarding the patient educational materials themselves and their dissemination will be reported. Conclusion: The data highlights differential engagement regarding the level of participation between the different stakeholders within the SCD community. It also reflects both similarities and differences with respect to satisfaction and perception of power dynamics in this process. With these results, we aim to introduce how a community-based dialogue approach can be used as a model for engagement for SCD clinical trials, for creation of stakeholder-informed patient education materials, and for other disease communities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gene Therapy Equity in Sickle Cell Disease: Distributional Cost-Effectiveness Analysis (DCEA) of Gene Therapy Vs. Standard-of-Care in Patients with Sickle Cell Disease in the United States

Introduction: Patients with sickle cell disease (SCD) face substantial mortality risks and decreased quality-of-life for every year lived with disease. Once approved in the United States (US), gene therapy treatment for patients with SCD would allow the possibility of lifelong disease remission and, unlike allotransplantation, do so without the concomitant risks of allotransplantation. While gene therapy has previously been projected to be cost-ineffective using traditional cost-effectiveness criteria in the US, this does not include a quantification of the health disparities present in the clinical care of patients with SCD. We sought to address this knowledge gap by conducting the first distributional cost-effectiveness analysis, a method that quantifies the tradeoffs between traditional cost-effectiveness outcomes and health equity, for gene therapy use in SCD. Methods: We built and validated a Markov simulation model of patients with a diagnosis of mild, moderate, or severe SCD to examine the cost-effectiveness of gene therapy versus patients treated with standard-of-care (SOC). Gender-, age-, and disease-severity-specific annual costs and transition probabilities between disease severities were informed by ten years of real-world data from commercially insured patients in the United States with SCD (2007-2017; Optum claims data). Disease severity was defined based on annual number of hospitalizations and SOC was inclusive of hydroxyurea, opioid therapy, antibiotics, vaccinations, blood transfusions, and stem cell transplantation. Quality-adjusted life-years (QALYs) were informed by published lifetime simulation of patients living with SCD and matched controls. These were originally derived from a polynomial fit linking Euroqual-5 Dimensions to the visual analog scale for pain in patients with SCD in several studies. Age-, gender- and disease-specific background mortality probabilities were employed. Traditional cost-effectiveness was quantified using an incremental cost-effectiveness ratio (ICER) and a cost-effectiveness threshold of $100,000/QALY. Base-case gene therapy was assumed to cost $2.1 million based on other recently approved indications, be 100% effective in achieving disease remission, and applied to a minimum eligibility age of 12, as studied in randomized, controlled trials. To account for health inequities in SCD we then conducted a distributional cost-effectiveness analysis (DCEA), which applies an inequality aversion parameter (i.e., equity weight) to the distribution of outcomes across relevant sub-groups. Specifically, we derived the threshold inequality aversion parameters needed for gene therapy to be favored over SOC and justify funding per DCEA standards. These were compared to prior estimates for commonly used equity weights in the US (range 0.5-3.0) and the United Kingdom (UK) (11.0-28.9). Results: Base-case gene therapy versus SOC treatment starting at age 12 yields 25.5 versus 16.0 discounted lifetime QALYs at costs of $2.4 and $1.1 million, respectively. The ICER of $144,000/QALY is not good value for money using traditional cost-effectiveness methods and a cost-effectiveness threshold of $100,000/QALY. When using DCEA that account for health disparities in SCD, the inequality aversion parameter threshold is 1.7. This implies a preference for reducing disparities in SCD care that is in line with prior estimates in the US and well below those of the UK, per DCEA standards to favor gene therapy over SOC. A corresponding price range for gene therapy for SCD across the range of commonly used equity weights in the US is $1.4-$3.0 million (Figure 1). Conclusion: Gene therapy can be an equitable therapeutic strategy for patients with SCD in the United States despite a projected minimum cost of $2.1 million once approved, based on commonly-used equity weight values used in DCEA. Equitable gene therapy use would include expanding eligibility to include patients with mild and moderate disease severity, in addition to those with severe SCD, assuming similar therapeutic efficacy of gene therapy across disease severity. Rather than relying on ranges of commonly-used equity weights, nationally representative surveys should be fielded to empirically deriving an inequality aversion parameter value for addressing this SCD-specific health disparity to further help inform an equitable price benchmark for gene therapy in the US. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Diverse Approaches to Gene Therapy of Sickle Cell Disease.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the β-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Clonal Hematopoiesis and the Risk of Hematologic Malignancies after Curative Therapies for Sickle Cell Disease.

Sickle cell disease (SCD) is associated with severe morbidity and early mortality. Two large population studies found an increased risk for leukemia in individuals with SCD. Notably, while the relative risk of leukemia development is high, the absolute risk is low in individuals with SCD who do not receive cell-based therapies. However, the risk of leukemia in SCD is high after graft rejection and with gene therapy. Clonal hematopoiesis (CH) is a well-recognized premalignant condition in the general population and in patients after high-dose myelotoxic therapies. Recent studies suggest that CH may be more common in SCD than in the general population, outside the cell-based therapy setting. Here, we review risk factors for CH and progression to leukemia in SCD. We surmise why patients with SCD are at an increased risk for CH and why leukemia incidence is unexpectedly high after graft rejection and gene therapy for SCD. Currently, we are unable to reliably assess genetic risk factors for leukemia development after curative therapies for SCD. Given our current knowledge, we recommend counseling patients about leukemia risk and discussing the importance of an individualized benefit/risk assessment that incorporates leukemia risk in patients undergoing curative therapies for SCD.

28-Year-Old Man With Joint Pain

28-Year-Old Man With Joint Pain

Current Sickle Cell Disease Gene Therapy Treatments: Literature Review

Sickle cell disease (SCD) consists of haemoglobin-mutation related blood disorders caused by mutations of the HBB gene. Current treatments for SCD are symptom-based or preventive treatments. The only curative treatment for SCD- an allogeneic hematopoietic stem cell transplant- is inaccessible to the majority of SCD patients. The transplant required a donor graft is unavailable for most individuals. Currently, research in gene therapy treatment for SCD attempts to provide long-lasting treatments in two distinct techniques. The first technique is to change the mutation-containing genotype to produce a normal or functional haemoglobin protein. The second technique is to bypass the production of the mutated adult haemoglobin and product fetal haemoglobin instead. This literature review compares three gene-editing methods; Lentiviral Vectors, CRISPR/Cas9, and Base Editors. A review of previously published research papers was conducted and compared over a 2 month period during a summer student research program to determine the progression of each of the three gene-editing methods in the two techniques of SCD gene therapy treatment. While all three were successful in both genotype correction and fetal haemoglobin induction, only the Lentiviral Vector and CRISPR/Cas9 treatments for fetal haemoglobin induction have published data on human trials. However, the Base Editor shows promise in its ability to surpass many issues faced with both viral vectors and CRISPR/Cas9 such as off-target DNA breaks. Progression in SCD gene therapy can provide a treatment option for all affected individuals and can even provide a basis for gene therapy for other blood disorders.

Steps to Fetal Gene Therapy: Disruption of Bcl11a erythroid-specific enhancer region with CRISPR/Cas9

The clinical severity and lifelong morbidity of sickle cell disease (SCD) and b-thalassemia, the major disorders of b-globin gene mutations, can be ameliorated by production of fetal hemoglobin (HbF). In fetal life, g-globin (encoded by 2 duplicated genes in the b-globin locus) does not use the mutated adult b-hemoglobin (HbA). The HbF to HbA transition is regulated by a repressor, Bcl11a, which silences fetal g-globin. Given our overarching goal to develop a fetal gene therapy for SCD which “unsilences” g-globin, we aimed to disrupt the Bcl11a erythroid-specific enhancer region using three different synthetic single guide RNA (sgRNA) sequences with CRISPR/Cas9 in murine erythroleukemia (MEL) cells (Fig.

Patient and Caregiver Attitudes Towards Gene Therapy for Sickle Cell Disease: A Need for Partnership and Education

Background:Fewer than 20% of patients with sickle cell disease (SCD) have access to curative matched sibling donor bone marrow transplantation (MSD-BMT). Development of novel therapies to ameliorate the serious morbidity and early mortality caused by SCD is a National Institute of Health research priority. Gene therapy for SCD has emerged as a promising approach in early-stage clinical trials approach with the potential to reach greater numbers of patients than MSD-BMT. Knowledge and attitudes of individuals with SCD towards this investigational new therapy and their willingness to participate in early-stage clinical trials have not been systematically described.Methods:Patients with SCD at least 13 years of age (n=66) and caregivers of children with SCD (n=38) were surveyed about knowledge, attitudes, and beliefs regarding gene therapy for SCD. Most questions utilized a 5-item Likert response scale while The Newest Vital Sign was used to assess health literacy. To explore attitudes around gene therapy in further detail, we identified a regionally diverse focus group comprised of 12 patients/caregivers with SCD from across the United States. We audio-recorded, transcribed verbatim, and analyzed five focus group discussions using thematic content analysis to identify salient themes related to attitudes towards gene therapy for SCD. Our goal was to identify the educational needs and preferences of this patient / caregiver stakeholder group.Results:Survey: Of the 104 survey respondents 96% identified as Black and 4% as Hispanic. Respondents were between 13-64 years (mean 29.6, Std Dev 11.9 years). HbSS and HbSC were the most common genotypes (61.5% and 24%, respectively). Only 4.8% of participants (N=5) felt “extremely knowledgeable” about gene therapy for SCD while most (63.4%) reported no/slight knowledge. We found no association between health literacy levels and gene therapy knowledge (p=0.361). Nearly 30% of participants reported that the risk of cancer as a potential side effect would “probably not prevent their enrollment” on a gene therapy trial while 48% said it “definitely/probably would preclude their participation”. Most respondents had a neutral attitude regarding the safety of gene therapy for SCD and how good of a treatment it was (56.7% and 58.6%, respectively). Only a few respondents endorsed the idea that gene therapy was “unsafe” or “not a good treatment for SCD” (5.8% and 4.8% respectively). There was an association between increasing knowledge about gene therapy and agreement that it is safe (p=0.012) and a good treatment for SCD (p=0.031).Focus Groups: Among the focus group participants, there was consensus that communication about gene therapy is suboptimal. Participants noted that clinicians frequently use medical jargon, do not tailor their approach to the individual patient/caregiver, and fail to adequately disclose important details. Focus group members desired more information about side effects and risks associated with conditioning chemotherapy, immunosuppression, risk of infertility, and hair loss. Participants, particularly parents of children with SCD, were worried about future unknown risks and described the enormity of the decision to enroll in an early-phase trial. Focus group members verbalized the need for inclusion of patients with SCD as partners during the informed consent/assent process and ideally, at the outset of the clinical trial design.Conclusion:Very few patients with SCD described feeling knowledgeable about gene therapy for SCD; a majority have neutral feelings about the safety and utility (“good treatment for SCD”) of this new approach. Patient/caregiver-centered education about gene therapy in a manner that meets stakeholder informational needs is urgently needed. Given the temporary hold on lentiviral gene therapy trials for SCD (after some participants developed a myeloid neoplasm on an industry sponsored trial in February 2021) and the risk of cancer as a probable or definite barrier to enrollment in a gene therapy trial for many patients, transparency of information about these risks is essential. For the successful execution of novel trials in SCD, a community-based participatory research approach is vital. DisclosuresSharma: Medexus Inc: Consultancy; Spotlight Therapeutics: Consultancy; Novartis: Other: Salary support paid to institution; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Vindico Medical Education: Honoraria; CRISPR Therapeutics: Other, Research Funding. Johnson: CRISPR Therapeutics: Research Funding.

Impact of Ethnic Origin on CRISPR/Cas Off-Target Prediction for Guide RNAs Used in Gene Therapy for Sickle Cell Disease and Other Genetic Diseases

Background: Although gene editing by CRISPR/Cas9 is a promising curative strategy for inherited and acquired diseases, potential off-targets remain a major concern. Most computational and biochemical methods for off-target search have focused on single genomes, not considering the impact of genetic diversity among human populations. Previous studies showed that genetic variants might significantly impact on- and off-target specificity. However, none of them explored the most extensive worldwide human genetic diversity dataset to examine the impact of population genetic variation on therapeutically relevant guide RNAs (gRNAs).Objective: To evaluate how genetic variation among human populations affects the predicted off-targets for gRNAs that have been used in clinical trials or had consistent results in preclinical studies.Methods: We selected 13 gRNAs tested for sickle cell disease (SCD), severe combined immunodeficiency, chronic granulomatous disease, X-linked hyper IgM syndrome, acquired immunodeficiency syndrome, and Leber congenital amaurosis. Off-target prediction was performed against the reference genome (hg38) and against population-specific genomes using CRISPRitz (https://github.com/pinellolab/CRISPRitz). Genomic variants with allele frequencies ≥1 % were obtained from both the entire gnomAD v3.0 dataset (76,156 whole-genome sequences) and each ethnic group separately. Off-target sequences were filtered considering up to four mismatches and bulges, and a cutting frequency determination (CFD) score ≥0.2 for those harbouring only mismatch events. Sequences were annotated with Annovar (https://annovar.openbioinformatics.org/).Results: A total of 1,727 off-target events were found against the reference genome for the 13 selected gRNAs. 134 additional events were detected with the gnomad_all dataset. When variants from different ethnic groups were considered 234 additional off-targets were observed. The distribution of CFD values was comparable between the population-specific/additional and the reference genome off-targets, as well as the distribution of their genomic localization. Most events mapped to intergenic regions (~50%), followed by intronic regions (~35%). The proportion of additional events varied for the different gRNAs (ranging from 7.8% to 27.7% increase), and the number of events was also variable among the populations. Considering the different ethnic groups, the highest number of events was found in African-ancestry populations (164) and the lowest in the non Finish European population (87), implying that some off-target events were found exclusively in one of the populations (91 in total). Although 219 (75%) of the variants within the population-specific/ additional off-targets have a frequency between 1-10%, 20 (6.8%) have a frequency higher than 80% in at least one of the populations analysed. Considering only the gRNAs used in gene therapy for SCD (n=6), 100 additional predicted off-targets were found in the African population of which 29 are exclusive, while only 77 were found in gnomad_all and 43 in the non-Finish European population, being 2 exclusive.Discussion: Rigorous analysis regarding off-target events is a necessary step to address the safety of gene therapy strategies. Although in silico analysis against the reference genome captures most predicted off-target events, we and others have demonstrated that an important proportion of additional events are lost if population-specific variants are not considered. Herein, we showed that the number of additional events is particularly higher when variants with allele frequencies >=1% in African populations are considered. This might occur because the African population is more genetically diverse and the reference genome was assembled based on Caucasian individuals. In addition, several off-target events are exclusively found in non-African populations, showing the importance of considering the genetic background of patients who undergo gene therapy. Additional caution should be taken for diseases that are more frequent in specific populations, such as SCD and β-thalassemia, for which there are several ongoing gene therapy trials. DisclosuresNo relevant conflicts of interest to declare.