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Lentiviral Gene Therapy with CD34+ Hematopoietic Cells for Hemophilia A. BACKGROUND: Severe hemophilia A is managed with factor VIII replacement or hemostatic products that stop or prevent bleeding. Data on gene therapy with hematopoietic stem-cell (HSC)-based expression of factor VIII for the treatment of severe hemophilia A are lacking. METHODS: We conducted a single-center study involving five participants 22 to 41 years of age with severe hemophilia A without factor VIII inhibitors. Autologous HSCs were transduced with CD68-ET3-LV - a lentiviral vector including a new F8 transgene (ET3) with a myeloid-directed CD68 promoter - either without transduction enhancer (group 1) or with transduction enhancer (group 2). Transduced HSCs were transplanted into recipients after myeloablative conditioning. The treatment was assessed for safety (engraftment and regimen-related toxic effects) and efficacy (factor VIII activity and annualized bleeding rate). RESULTS: Participants received CD68-ET3-LV-transduced autologous CD34+ HSCs at doses of 5.0×106 to 6.1×106 per kilogram of body weight. The vector copy numbers in the final drug product were 1.0 and 0.6 copies per cell for the two participants in group 1 and 1.5, 0.6, and 2.2 copies per cell for the three participants in group 2. The duration of severe neutropenia was 7 to 11 days and of severe thrombocytopenia was 1 to 7 days. The median factor VIII activity level, measured with the use of a one-stage assay, after day 28 until the last follow-up visit was 5.2 IU per deciliter (range, 3.0 to 8.7) and 1.7 IU per deciliter (range, 1.0 to 4.0) with a peripheral-blood vector copy number of 0.2 and 0.1 copies per cell, respectively, in the two group 1 participants, and 37.1 IU per deciliter (range, 18.3 to 73.6), 19.3 IU per deciliter (range, 6.6 to 34.5), and 39.9 IU per deciliter (range, 20.6 to 55.1) with a peripheral-blood vector copy number of 4.4, 3.2, and 4.8 copies per cell, respectively, in the three group 2 participants. The annualized bleeding rate was zero for all five participants over a cumulative follow-up of 81 months (median follow-up, 14 months; range, 9 to 27). CONCLUSIONS: Gene therapy for hemophilia A with the use of lentiviral vector-transduced autologous HSCs resulted in stable factor VIII expression, with factor VIII activity correlating to vector copy number in the peripheral blood. (Funded by the Ministry of Science and Technology, Government of India, and others; ClinicalTrials.gov number, NCT05265767; Clinical Trials Registry-India number, CTRI/2022/03/041304.).

Severe hemophilia A is an X-linked disorder characterized by less than 1% measurable Factor VIII (FVIII) activity in plasma and is associated with frequent spontaneous bleeding into joints and muscles as well as sporadic life-threatening bleeding. (1). People with hemophilia (PWH) with FVIII activity >1 international unit (IU) per deciliter (dL) have a milder clinical profile while among those with FVIII activity >5 IU per dL, spontaneous bleeding is rare. (1) Prophylactic treatment with FVIII products or other hemostasis drugs are recommended for prevention of bleeding in severe hemophilia. (1) Gene therapy for hemophilia has been attempted for more than two decades with different technologies.(2–4) An adeno-associated virus (AAV) vector-based hepatocyte directed gene therapy has been approved for the treatment of hemophilia A. (4) However, this technology has several limitations including i) variable initial expression; ii) gradual decline of expression after the first 6–12 months, often associated with transaminitis requiring immunosuppressive treatment, which has led to its own adverse events (4, 5); iii) age-related ineligibility until liver maturity (6); and iv) pre-existing anti-AAV antibodies from natural infections, precluding treatment. (7, 8)

ne of expression after the first 6–12 months, often associated with transaminitis requiring immunosuppressive treatment, which has led to its own adverse events (4, 5); iii) age-related ineligibility until liver maturity (6); and iv) pre-existing anti-AAV antibodies from natural infections, precluding treatment. (7, 8) We have shown it is possible to achieve FVIII expression in hemophilia A mice using mouse hematopoietic stem cells (HSC) and in immunodeficient mice using human CD34+ HSC with a CD68-ET3-LV self-inactivating lentiviral vector packaging a novel F8 transgene (ET3). (9) These preclinical data led us to initiate a first-in-human clinical trial to evaluate the safety and feasibility of this technology for gene therapy of severe hemophilia A. This report describes the outcome of the completed phase 1 clinical trial involving five participants who have completed at least 6 months of follow-up.

This is a single-center study involving five participants with severe hemophilia A without inhibitors who have undergone gene therapy by transplantation of autologous HSC transduced with the CD68-ET3-LV vector. The study was approved by the Institutional Review Board and the Ethical Committee of the Christian Medical College, Vellore, India as well as the Central Drugs Standard Control Organization (CDSCO) of the Ministry of Health and Family Welfare of Government of India and was conducted according to GCP guidelines. The investigators at the Christian Medical College (CMC), Vellore, India and the Centre for Stem Cell Research (CSCR), a unit of inStem, Bengaluru, India along with collaborators at the Emory University, Atlanta, GA, USA and Expression Therapeutics, Tucker, GA, USA were responsible for design of this study and having the CD68-ET3-LV vector manufactured under suitable GMP conditions. The study was conducted at the CMC and CSCR, Vellore, India where site investigators and team members were responsible for collection and analysis of all data. The manuscript was prepared by the lead authors and reviewed by all authors who also had access to the complete data. The authors assure the completeness and accuracy of the data and compliance of the study with the protocol which is available at NEJM.org.

mbers were responsible for collection and analysis of all data. The manuscript was prepared by the lead authors and reviewed by all authors who also had access to the complete data. The authors assure the completeness and accuracy of the data and compliance of the study with the protocol which is available at NEJM.org. As a first of its kind study in India, a multistep process was followed for participant selection and informed consent. This started with cohort counselling for groups of people with hemophilia over an extended period to increase general awareness in the community about gene therapy technologies. After approval of the clinical trial protocol, specific information was shared with the same patient groups. Interested patients and their families were invited for further discussion to the study site following which detailed counselling was undertaken for the potential participant and his family. Formal consenting was video recorded according to regulatory requirements in India. Going beyond these regulatory requirements, an additional step of review by an independent external committee comprising of a physician, a family member of an unrelated person with hemophilia (PWH) and an HSC transplant survivor was included. This committee interviewed the potential participant and his family to evaluate their comprehension of this clinical trial and, if satisfied with their responses, certified the consenting process.

g of a physician, a family member of an unrelated person with hemophilia (PWH) and an HSC transplant survivor was included. This committee interviewed the potential participant and his family to evaluate their comprehension of this clinical trial and, if satisfied with their responses, certified the consenting process. A total of eight participants were consented. Two participants could not be included in the study, one due to failed eligibility criteria and the second who refused participation due to concerns of possible infertility. The third participant was withdrawn when he developed a low titer FVIII inhibitor on the day his conditioning treatment was to start. His outcome will be reported separately. Data from the other five treated participants treated between June, 2022 and December, 2023 are included in this report. (Table S1) Study participants, who were all on episodic treatment, received prophylactic extended half-life (EHL) recombinant FVIII (rFVIII) once or twice weekly after consenting to the study. CD34+ cells were mobilized with granulocyte colony-stimulating factor (G-CSF) at 5ug per kg given twice daily for 5 days along with plerixafor 0.24mg/kg given 12–14 hours before apheresis. Adequate numbers of autologous CD34+ peripheral blood stem cells (PBSC), defined as >5×106 CD34+ cells/kg body weight, were obtained in a single apheresis procedure after positive selection using a CliniMACS® Plus system (Miltenyi Biotec, Bergisch Gladbach, Germany). An aliquot of PBSC with >2×106/kg CD34+ cells per kg was cryo-preserved without manipulation.

pheral blood stem cells (PBSC), defined as >5×106 CD34+ cells/kg body weight, were obtained in a single apheresis procedure after positive selection using a CliniMACS® Plus system (Miltenyi Biotec, Bergisch Gladbach, Germany). An aliquot of PBSC with >2×106/kg CD34+ cells per kg was cryo-preserved without manipulation. The enriched CD34+ HSC were transduced with the clinical grade lentiviral vector, CD68-ET3-LV, (Figure S1) initially using a double exposure transduction protocol. (Suppl P1) After the outcome of the first two treated participants was reviewed, and as planned, the transduction protocol was modified to include an enhancer, (LentiBOOST, SIRION Biotech, Gräfelfing, Germany), and only a single exposure to the vector. Transduced HSCs were cryopreserved while the final drug product was qualified for release criteria. Participants were conditioned with intravenous treosulfan at 14g/m2 on days −6 to −4 along with fludarabine 30mg/m2 on days −6 to −2 with no therapeutic drug monitoring. The day of infusion of the drug product was considered as day 0. Participants were nursed in HEPA-filtered isolation rooms during conditioning and ensuing cytopenia. Other supportive care was provided according to institutional protocols for autologous HSC transplantation (HSCT).

−2 with no therapeutic drug monitoring. The day of infusion of the drug product was considered as day 0. Participants were nursed in HEPA-filtered isolation rooms during conditioning and ensuing cytopenia. Other supportive care was provided according to institutional protocols for autologous HSC transplantation (HSCT). The endpoints related to safety included i) adverse events related to drug-product infusion and conditioning therapy, ii) engraftment by day +30, iii) survival at day +100 and iv) development of a FVIII inhibitor v) vector copy number and vector integration profile. The endpoints of efficacy were i) plasma FVIII activity (one stage assay – OSA and chromogenic substrate assay – CSA) and ii) annualized bleeding rate (ABR). (Table S2) Participants were assessed regularly for adverse events according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (2017). The engraftment endpoint criteria were absolute neutrophil count of >0.5×109 per liter and an unsupported platelet count of >20×109 per liter. FVIII activity assay was performed daily during the conditioning and cytopenic phase to maintain trough levels >50 IU per dL after administration of EHL rFVIII. As engraftment ensued, the dose of EHL rFVIII was gradually reduced and stopped once the platelet count was >100×109 per liter.

let count of >20×109 per liter. FVIII activity assay was performed daily during the conditioning and cytopenic phase to maintain trough levels >50 IU per dL after administration of EHL rFVIII. As engraftment ensued, the dose of EHL rFVIII was gradually reduced and stopped once the platelet count was >100×109 per liter. Vector copy number (VCN) in the drug product and in genomic DNA from peripheral blood post-gene therapy was assessed at regular intervals, as described previously. (9) (Suppl P2) In view of the underlying hemophilia, bone marrow assessment was performed on one occasion under additional FVIII cover at 4–22 months of follow-up for assessment of morphology, cytogenetics and VCN. Integration site analysis was performed by ligation mediated PCR (LM-PCR) on genomic DNA from peripheral blood and bone marrow (CD Genomics, New York, USA). The study was performed according to protocol approved by the regulator in India, the CDSCO. Serious adverse events were reported within 24 hours. An institutional data and safety monitoring board also reviewed serious adverse events. Additional scientific oversight was provided by an international scientific advisory board of experts. The data reported are as of 30th September, 2024. Given the small number of participants in this study, all statements are qualitative based on inspection of data with no statistical analysis.

As a first of its kind study in India, a multistep process was followed for participant selection and informed consent. This started with cohort counselling for groups of people with hemophilia over an extended period to increase general awareness in the community about gene therapy technologies. After approval of the clinical trial protocol, specific information was shared with the same patient groups. Interested patients and their families were invited for further discussion to the study site following which detailed counselling was undertaken for the potential participant and his family. Formal consenting was video recorded according to regulatory requirements in India. Going beyond these regulatory requirements, an additional step of review by an independent external committee comprising of a physician, a family member of an unrelated person with hemophilia (PWH) and an HSC transplant survivor was included. This committee interviewed the potential participant and his family to evaluate their comprehension of this clinical trial and, if satisfied with their responses, certified the consenting process.

The endpoints related to safety included i) adverse events related to drug-product infusion and conditioning therapy, ii) engraftment by day +30, iii) survival at day +100 and iv) development of a FVIII inhibitor v) vector copy number and vector integration profile. The endpoints of efficacy were i) plasma FVIII activity (one stage assay – OSA and chromogenic substrate assay – CSA) and ii) annualized bleeding rate (ABR). (Table S2) Participants were assessed regularly for adverse events according to the Common Terminology Criteria for Adverse Events (CTCAE) v5.0 (2017). The engraftment endpoint criteria were absolute neutrophil count of >0.5×109 per liter and an unsupported platelet count of >20×109 per liter. FVIII activity assay was performed daily during the conditioning and cytopenic phase to maintain trough levels >50 IU per dL after administration of EHL rFVIII. As engraftment ensued, the dose of EHL rFVIII was gradually reduced and stopped once the platelet count was >100×109 per liter. Vector copy number (VCN) in the drug product and in genomic DNA from peripheral blood post-gene therapy was assessed at regular intervals, as described previously. (9) (Suppl P2) In view of the underlying hemophilia, bone marrow assessment was performed on one occasion under additional FVIII cover at 4–22 months of follow-up for assessment of morphology, cytogenetics and VCN. Integration site analysis was performed by ligation mediated PCR (LM-PCR) on genomic DNA from peripheral blood and bone marrow (CD Genomics, New York, USA).

The study was performed according to protocol approved by the regulator in India, the CDSCO. Serious adverse events were reported within 24 hours. An institutional data and safety monitoring board also reviewed serious adverse events. Additional scientific oversight was provided by an international scientific advisory board of experts. The data reported are as of 30th September, 2024. Given the small number of participants in this study, all statements are qualitative based on inspection of data with no statistical analysis.

Five participants, aged 22–41 years at study recruitment, received the investigational gene therapy product candidate. The median follow-up is 14 months (range, 9–27) after gene modified HSC transplantation. The target number of mobilized CD34+ HSC was collected in a single apheresis yielding a median of 7.5×106 cells per kg body weight (range, 7.0–9.2). (Table S3) This also allowed a minimum of 2×106/kg unmanipulated CD34+ HSCs to be cryopreserved as a back-up, if required, for delayed engraftment. The median drug product dose infused after transduction was 5.6×106 cells per kg (range, 5.0–6.1). (Table 1) For the first two participants (G1) where the final gene therapy product, CD68-ET3-LV-CD34+, was manufactured with a double transduction protocol, the VCN was 0.57 and 1.02 in the drug product. For the next three participants, with the modified transduction protocol with a single transduction step with an enhancer, LentiBOOST®, the VCN in the drug product was 0.62, 1.49 and 2.24. (Table 1)

, CD68-ET3-LV-CD34+, was manufactured with a double transduction protocol, the VCN was 0.57 and 1.02 in the drug product. For the next three participants, with the modified transduction protocol with a single transduction step with an enhancer, LentiBOOST®, the VCN in the drug product was 0.62, 1.49 and 2.24. (Table 1) All participants developed expected severe cytopenia with nadir absolute neutrophil counts <0.1×109 per liter and platelet counts <20×109 per liter. However, engraftment occurred in all participants with the median time to neutrophil engraftment of 11 days (range, 10–12) and platelet engraftment of 15 days (range, 12–15). (Table S4) All participants received G-CSF 5ug per kilogram per day from day +5 after drug product infusion. The median duration of severe neutropenia was 8 days (range, 7–11) and that of severe thrombocytopenia was 3 days (range, 1–7). Engraftment parameters of participants were not different in G1 or G2 groups. The blood counts have been in the normal range for all participants between 1–3 months after gene therapy and in P6 by the 14th month. (Table S5). Exogenous EHL FVIII concentrate was administered for a median of 16 days (range: 11–20) during HSCT. No other adverse events of grade >2 occurred in any participant. The most common adverse event was nausea with or without vomiting. All non-laboratory adverse events during the hospital stay and follow-up are listed in Table S6A,B. No participant developed inhibitors to FVIII after drug product infusion.

All participants developed expected severe cytopenia with nadir absolute neutrophil counts <0.1×109 per liter and platelet counts <20×109 per liter. However, engraftment occurred in all participants with the median time to neutrophil engraftment of 11 days (range, 10–12) and platelet engraftment of 15 days (range, 12–15). (Table S4) All participants received G-CSF 5ug per kilogram per day from day +5 after drug product infusion. The median duration of severe neutropenia was 8 days (range, 7–11) and that of severe thrombocytopenia was 3 days (range, 1–7). Engraftment parameters of participants were not different in G1 or G2 groups. The blood counts have been in the normal range for all participants between 1–3 months after gene therapy and in P6 by the 14th month. (Table S5). Exogenous EHL FVIII concentrate was administered for a median of 16 days (range: 11–20) during HSCT. No other adverse events of grade >2 occurred in any participant. The most common adverse event was nausea with or without vomiting. All non-laboratory adverse events during the hospital stay and follow-up are listed in Table S6A,B. No participant developed inhibitors to FVIII after drug product infusion. Integration site analysis performed at 4–22 months after drug-product infusion has shown no safety concerns. Integration is skewed toward intronic sequences which account for >70% of integrations (Table S7 A, B, C), with exonic sequences accounting for <3% integrations and the rest being predominantly in the intergenic non-coding regions. (Table S7D) The current data indicate that the transduction enhancer does not adversely affect the integration profile of this LV vector. Though integrations were observed near several oncogenic sequences (Table S7E), consistent with other LV vector therapies (10–12), no evidence of clonal dominance was noted in any participant.

) The current data indicate that the transduction enhancer does not adversely affect the integration profile of this LV vector. Though integrations were observed near several oncogenic sequences (Table S7E), consistent with other LV vector therapies (10–12), no evidence of clonal dominance was noted in any participant. Bone marrow examination performed at 4–22 months after gene therapy for all participants, showed normal cellularity and morphology. Karyotype analysis on the bone marrow was also normal. Analysis of VCN on the bone marrow sample and the peripheral blood taken on the same day was comparable. (Table 2) Evidence of endogenous FVIII expression was observed as early as day +18 in P6. The FVIII activity (IU per dL) on day +60 was 7.9 and 4.0 in G1 participants and 28, 14.2 and 36.6 in G2 participants. Median FVIII activity (IU per dL) after day +28 until last follow-up are 5.2 (range, 3.0–8.7) and 1.7(range, 1.0–4.0) in the G1 participants and 37(range, 18.3–73.6), 19.3 (range, 6.6–34.5) and 39.9(range, 20.6–55.1-) in the G2 participants. All these FVIII levels are based on the OSA. The serial FVIII activities are shown in Figure 1 and Table S8. It is important to note that both the OSA and CSA FVIII activity showed similar results. (Table 2; Figure S2) No spontaneous bleeding events occurred in any participant during HSCT or after gene therapy. Before gene therapy, all participants had reported an ABR of >20 events. (Table 1) The lack of bleeding in P3 with FVIII activity between 1–3 IU per dL is notable.

Evidence of endogenous FVIII expression was observed as early as day +18 in P6. The FVIII activity (IU per dL) on day +60 was 7.9 and 4.0 in G1 participants and 28, 14.2 and 36.6 in G2 participants. Median FVIII activity (IU per dL) after day +28 until last follow-up are 5.2 (range, 3.0–8.7) and 1.7(range, 1.0–4.0) in the G1 participants and 37(range, 18.3–73.6), 19.3 (range, 6.6–34.5) and 39.9(range, 20.6–55.1-) in the G2 participants. All these FVIII levels are based on the OSA. The serial FVIII activities are shown in Figure 1 and Table S8. It is important to note that both the OSA and CSA FVIII activity showed similar results. (Table 2; Figure S2) No spontaneous bleeding events occurred in any participant during HSCT or after gene therapy. Before gene therapy, all participants had reported an ABR of >20 events. (Table 1) The lack of bleeding in P3 with FVIII activity between 1–3 IU per dL is notable. A positive correlation was noted between the VCN in peripheral blood 1 month after gene therapy and the corresponding FVIII activity. VCN analysis of peripheral blood and bone marrow also correlated well in both G1 and G2 participants. Both VCN and FVIII activity are much higher in G2 participants after HSCT (Table 2) even though the VCN in the gene therapy products were similar. This increase in FVIII activity also correlates with a more intense integration profile of the vector in the G2 participants. (Table S7B)

ell in both G1 and G2 participants. Both VCN and FVIII activity are much higher in G2 participants after HSCT (Table 2) even though the VCN in the gene therapy products were similar. This increase in FVIII activity also correlates with a more intense integration profile of the vector in the G2 participants. (Table S7B) All participants reported higher physical activities without fear of bleeding and enhanced ability to engage in more intense leisure activities. Two participants from G2 had trauma. P6 (FVIII activity of about 40 IU per dL) had a minor motor vehicle accident (MVA) on day +302 with minor cuts that stopped bleeding spontaneously and transient loss of consciousness but full neurological recovery immediately thereafter. CT scan of the brain was normal. P7 (FVIII activity of about 20 IU per dL) had a superficial cat bite on day +102 for which anti-rabies vaccine was given intramuscularly and a minor MVA on day +179 with superficial abrasions. No additional FVIII replacement was required for any of these trauma related events. Semen analysis carried out on all participants at least 6 months after HSCT showed normal sperm counts in four (one with low motility) and the fifth having a low sperm count at 8 months of follow-up.

All participants developed expected severe cytopenia with nadir absolute neutrophil counts <0.1×109 per liter and platelet counts <20×109 per liter. However, engraftment occurred in all participants with the median time to neutrophil engraftment of 11 days (range, 10–12) and platelet engraftment of 15 days (range, 12–15). (Table S4) All participants received G-CSF 5ug per kilogram per day from day +5 after drug product infusion. The median duration of severe neutropenia was 8 days (range, 7–11) and that of severe thrombocytopenia was 3 days (range, 1–7). Engraftment parameters of participants were not different in G1 or G2 groups. The blood counts have been in the normal range for all participants between 1–3 months after gene therapy and in P6 by the 14th month. (Table S5). Exogenous EHL FVIII concentrate was administered for a median of 16 days (range: 11–20) during HSCT. No other adverse events of grade >2 occurred in any participant. The most common adverse event was nausea with or without vomiting. All non-laboratory adverse events during the hospital stay and follow-up are listed in Table S6A,B. No participant developed inhibitors to FVIII after drug product infusion.

) The current data indicate that the transduction enhancer does not adversely affect the integration profile of this LV vector. Though integrations were observed near several oncogenic sequences (Table S7E), consistent with other LV vector therapies (10–12), no evidence of clonal dominance was noted in any participant. Bone marrow examination performed at 4–22 months after gene therapy for all participants, showed normal cellularity and morphology. Karyotype analysis on the bone marrow was also normal. Analysis of VCN on the bone marrow sample and the peripheral blood taken on the same day was comparable. (Table 2)

All participants reported higher physical activities without fear of bleeding and enhanced ability to engage in more intense leisure activities. Two participants from G2 had trauma. P6 (FVIII activity of about 40 IU per dL) had a minor motor vehicle accident (MVA) on day +302 with minor cuts that stopped bleeding spontaneously and transient loss of consciousness but full neurological recovery immediately thereafter. CT scan of the brain was normal. P7 (FVIII activity of about 20 IU per dL) had a superficial cat bite on day +102 for which anti-rabies vaccine was given intramuscularly and a minor MVA on day +179 with superficial abrasions. No additional FVIII replacement was required for any of these trauma related events. Semen analysis carried out on all participants at least 6 months after HSCT showed normal sperm counts in four (one with low motility) and the fifth having a low sperm count at 8 months of follow-up.

The results from this clinical trial validate a novel approach to gene therapy of severe hemophilia A through transplantation after myeloablative conditioning of autologous HSCs transduced with a LV vector encoding a novel FVIII transgene driven by a CD68 promoter. No unexpected safety issues were encountered. All participants achieved clinically significant circulating FVIII activity sufficient to completely abrogate spontaneous bleeding and which remained stable after the first 3–6 months during follow-up ranging from 9–27 months. The candidate CD68-ET3-LV CD34+ gene therapy product in this study incorporates multiple design elements that distinguish it from existing options. First, the ex vivo gene transfer into adult stem cells utilizing an integrating and self-inactivating LV vector instead of in vivo transfer to a hepatocyte through a non-integrating AAV vector. The correlative premise is lifelong durability of expression and the initial data out to more than two years of follow-up appear promising. Second, the human CD68 gene directed promoter shifts gene expression away from HSCs to prevent FVIII related cellular toxicity. (13, 14).

cyte through a non-integrating AAV vector. The correlative premise is lifelong durability of expression and the initial data out to more than two years of follow-up appear promising. Second, the human CD68 gene directed promoter shifts gene expression away from HSCs to prevent FVIII related cellular toxicity. (13, 14). Monocytes are abundant short lived circulating blood cells. (15) Tissue monocytes or macrophages also represent a second source of FVIII and can be long-lived (up to 1 yr) with ~ 200 billion macrophages present in an adult male. (16) Macrophages are also present at significant density in the synovium of joints and could contribute to local hemostasis even with low plasma FVIII activity. The third unique design element is the ET3 transgene, which results in 10 – 100-fold higher expression and secretion efficiency compared to B domain deleted human FVIII (17). The addition of a transduction enhancer in manufacturing produced a dramatic 5–10-fold increase in FVIII activity with the expected higher numbers of integration sites without affecting short term safety. The FVIII activity has been stable within the same clinically relevant range in all participants with some fluctuation during follow-up of >2 years in GI and >1 year in G2 participants. This could be related to analytical issues or reflect true variations in expression due to transient changes in the relevant blood cells during intercurrent events in the participants which were not clinically obvious.

participants with some fluctuation during follow-up of >2 years in GI and >1 year in G2 participants. This could be related to analytical issues or reflect true variations in expression due to transient changes in the relevant blood cells during intercurrent events in the participants which were not clinically obvious. Compared to AAV gene therapy, LV vector transduced HSC based gene therapy for hemophilia A overcomes nearly all the limitations related to highly variable and unpredictable expression, age and pre-existing anti-AAV antibody related ineligibilities. Interestingly, concordance between the OSA and CSA assays for FVIII activity is observed. However, the HSC approach also has limitations, including the elaborate and complex process of collection and manipulation of HSCs to prepare the patient-specific drug product. This approach has been used extensively for gene therapy of primary immune deficiency syndromes and also for the major hemoglobin disorders. (18, 19) Acute and long-term toxicities of myeloablative conditioning are also a potential concern (20), including severe mucositis and possible infertility. (21) Our results so far have been encouraging in both types of adverse events. Using treosulfan instead of busulfan, as in other myeloablative conditioning protocols for HSC based gene therapy, (18, 22) we show a much-reduced toxicity profile, as is well-known in allogeneic HSC transplant for high-risk cases. (23). What is most remarkable is the finding of normal sperm counts in four of the five participants in this study. Lower infertility rates have been reported with treosulfan conditioning compared to busulfan. (24) Finally, in severe genetic disorders, gene therapy should ideally be offered as soon as possible after diagnosis. This HSC-based approach moves the field closer to that goal. Treatment in early childhood well before puberty would also reduce infertility concerns (24, 25). Safety concerns related to random integration of the LV vector into the host genome will need close monitoring.

y be offered as soon as possible after diagnosis. This HSC-based approach moves the field closer to that goal. Treatment in early childhood well before puberty would also reduce infertility concerns (24, 25). Safety concerns related to random integration of the LV vector into the host genome will need close monitoring. In conclusion, the early results of this clinical trial reveal a new opportunity for gene therapy of hemophilia A that can be offered to all patients, possibly at an early age, and which results in sustained expression of therapeutic levels of FVIII. Longer follow-up and additional clinical trials with more participants will clarify whether these expectations will hold true.