• ClinicalTrials.gov Identifier: NCT05265767¹
• Phase 1, Single Group Assignment, open label trial¹

The status of the trial was reported on ClinicalTrials.gov¹ as follows

• Completed
• Last Update Posted: 2024-07-12

The key inclusion criteria were extracted from ClinicalTrials.gov¹ and are summarized as follows:

• Male subjects who are ≥18 years of age and < 45 years of age
• Diagnosis of severe hemophilia A (<1 IU/dL FVIII activity)
• Documented history of more than 100 exposures of factor VIII treatment
• Average of at least 3 bleeds requiring treatment per year over the prior three years,
  • at least 3 bleeds per year during the 3 years preceding the initiation of prophylaxis, or
  • evidence of joint damage (knee, elbow or ankle) on physical or
  • radiographic examination thought to be related to hemophilia

To view all inclusion criteria, refer to Identifier NCT05265767¹ on ClinicalTrials.gov

 The key exclusion criteria were extracted from ClinicalTrials.gov¹ and are summarized as follows:

• History of spontaneous central nervous system bleeding within the last 5 years.
• Subjects who have had prior cellular based therapy or gene editing/ gene therapy including a previous stem cell transplant
• History of a FVIII inhibitor (>0.6 Bethesda Units/ml) including at least 2 measurements over the preceding 5 years or any single titer >5 Bethesda Units (BU) /ml
• Previous stem cell transplant

To view all exclusion criteria, refer to Identifier NCT05265767¹ on ClinicalTrials.gov

Information regarding the vector manufacturing platform was collected from the protocol associated with reference #2 and is summarized as follows:

• The pseudotyping of the lentiviral vector was not reported
• CD68ET3-LV lentiviral vector particles were harvested from the supernatant of transfected 293T cells and purified via ion exchange chromatography
• CD34+ enriched hematopoietic stem cells (HSCs) were transduced with the CD68ET3-LV lentiviral vector

Hybrid human/porcine B-domain deleted FVIII variant (ET3, HP47) ^{2, 3, 4, 5}

The expression cassette includes a CD68 promoter to drive myeloid-directed gene expression²

Gene delivery was carried out by transplanting autologous CD34+ enriched HSCs transduced with the CD68-ET3-LV vector²

A list of all consented patients was taken from Table S1 in reference #2 and is summarized below

As reported in reference #2, the recipients of autologous HSCs were divided into two groups based on whether transduction with CD68-LV-ET3 was performed with or without a transduction enhancer.
The details considering the drug product dose are summarized in the table below:

Follow-up durations for each patient were collected from Table 1 in reference #2 and are summarized as follows:

As reported in reference #2, both OS and CH assays were used to assess FVIII activity

Data on FVIII activity relative to the vector copy number in peripheral blood and bone marrow was collected from Table 2 in reference #2 and is summarized as follows:

    † FVIII activity levels at the last follow-up time point were collected from Table S8 in reference #2

As shown in Figure 1 in reference #2, the times to peak response are summarized in the table below:

    † Peak responses were not reached until the 13-month follow-up time point

Data on annualized bleeding rates before and after gene therapy were extracted from Figure 1B in reference #2 and are summarized below in the table

The following statements on participants bleeding rates were collected from reference #2 as follows:

• No spontaneous bleeding events occurred in any participant during HSCT or after gene therapy
• Before gene therapy, all participants had reported an annualized bleeding rate of at least 20 events
• The lack of bleeding after gene therapy in Participant 3, who continued to have a factor VIII activity level of 1 to 3 IU per deciliter, is notable

• Annualized FVIII infusion rates or FVIII usage were not explicitly reported in reference #2 for the follow-up period after gene therapy.
• As reported in reference #2, two Group 2 participants who experienced trauma-related bleeding events did not require additional FVIII replacement therapy:
  • P6: Minor motor vehicle accident on day 302 with minor cuts that stopped bleeding spontaneously and an FVIII activity level of approximately 40 IU/dL
  • P7: Superficial cat bite on day 102 (received an intramuscular antirabies vaccine) and a minor motor vehicle accident with superficial abrasions on day 179, with FVIII activity levels of approximately 20 IU/dL at both time points

IRRs were not explicitly reported in reference #2

The following statements regarding AEs in treated participants were extracted from reference #2  and are summarized below:

• Except for neutropenia and thrombocytopenia, no other AEs greater than grade 2 occurred in any participant
• The most common adverse event was nausea, with or without vomiting
• Inhibitors to factor VIII did not develop in any participant after drug-product infusion

A detailed summary of AEs related to laboratory investigations is presented in Table S6B in reference #2 and shown in the table below:

To view non-laboratory investigation-related AEs, refer to Table S6 A in the supplementary appendix of reference #2

Nor reported

Elevated ALT levels, a marker of liver toxicity, are not a critical parameter for gene therapy involving the transplantation of gene-modified hematopoietic stem cells

Elevated AST levels, a marker of liver toxicity, are not a critical parameter for gene therapy involving the transplantation of gene-modified hematopoietic stem cells

Not applicable to gene therapy involving the transplantation of gene-modified hematopoietic stem cells

Not applicable to gene therapy involving the transplantation of gene-modified hematopoietic stem cells

Not applicable to gene therapy involving the transplantation of gene-modified hematopoietic stem cells

Statements on outcomes from critical safety assessments confirming that the gene therapy does not induce malignancies were taken from reference #2 and are summarized below

• Integration-site analysis performed at 4 to 22 months after drug-product infusion showed no safety concerns.
• Integration is skewed toward intronic sequences,
  • which account for more than 70% of integrations,
  • with exonic sequences accounting for less than 3% of integrations and
  • the rest being predominantly in the intergenic noncoding regions (Table S7)²
• The current data indicate that the transduction enhancer does not adversely affect the integration profile of this lentiviral vector.
• Although integrations were observed near several oncogenic sequences, which is consistent with other lentiviral vector therapies,^{6, 7, 8}
  • no evidence of clonal dominance was noted in any participant.
• Bone marrow examination performed at 4 to 22 months after gene therapy in all participants showed normal cellularity and morphology.
• Karyotype analysis of the bone marrow was also normal.
• Results of analyses of the vector copy number in samples of bone marrow and peripheral blood obtained on the same day were similar
  • (view table in the variable Mean Transgene Activity, % or IU/dL)

1. Hematopoietic Stem Cell Transplantation Gene Therapy for Treatment of Severe Hemophilia A. Available at: Researcher View | Hematopoietic Stem Cell Transplantation Gene Therapy for Treatment of Severe Hemophilia A | ClinicalTrials.gov
2. Srivastava A, Abraham A, Aboobacker F, Singh G, Geevar T, Kulkarni U, Selvarajan S, Korula A, Dave RG, Shankar M, Singh AS, Jeba A, Kumaar N, Benjamin C, Lakshmi KM, Srivastava VM, Shaji RV, Nair SC, Brown HC, Denning G, Lollar P, Doering CB, Spencer T. Lentiviral Gene Therapy with CD34+ Hematopoietic Cells for Hemophilia A. N Engl J Med. 2024 Dec 9. doi: 10.1056/NEJMoa2410597. Epub ahead of print. PMID: 39655790. Lentiviral Gene Therapy with CD34+ Hematopoietic Cells for Hemophilia A - PubMed
3. Brown, H.C., et al., Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery. Mol Ther Methods Clin Dev, 2014. 1: p. 14036. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery - PubMed (nih.gov)
4. Doering, C.B., et al., Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion. J Biol Chem, 2004. 279(8): p. 6546-52. 37. Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion* - Journal of Biological Chemistry (jbc.org)
5. Doering, C.B., et al., Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A. Hum Gene Ther, 2018. 29(10): p. 1183-1201. Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A - PMC (nih.gov)
6. Cowan MJ, Yu J, Facchino J, Fraser-Browne C, Sanford U, Kawahara M, Dara J, Long-Boyle J, Oh J, Chan W, Chag S, Broderick L, Chellapandian D, Decaluwe H, Golski C, Hu D, Kuo CY, Miller HK, Petrovic A, Currier R, Hilton JF, Punwani D, Dvorak CC, Malech HL, McIvor RS, Puck JM. Lentiviral Gene Therapy for Artemis-Deficient SCID. N Engl J Med. 2022 Dec 22;387(25):2344-2355. doi: 10.1056/NEJMoa2206575. PMID: 36546626; PMCID: PMC9884487. Lentiviral Gene Therapy for Artemis-Deficient SCID - PubMed
7. Yan KK, Condori J, Ma Z, Metais JY, Ju B, Ding L, Dhungana Y, Palmer LE, Langfitt DM, Ferrara F, Throm R, Shi H, Risch I, Bhatara S, Shaner B, Lockey TD, Talleur AC, Easton J, Meagher MM, Puck JM, Cowan MJ, Zhou S, Mamcarz E, Gottschalk S, Yu J. Integrome signatures of lentiviral gene therapy for SCID-X1 patients. Sci Adv. 2023 Oct 6;9(40):eadg9959. doi: 10.1126/sciadv.adg9959. Epub 2023 Oct 6. PMID: 37801507; PMCID: PMC10558130. Integrome signatures of lentiviral gene therapy for SCID-X1 patients - PubMed
8. Magnani A, Semeraro M, Adam F, Booth C, Dupré L, Morris EC, Gabrion A, Roudaut C, Borgel D, Toubert A, Clave E, Abdo C, Gorochov G, Petermann R, Guiot M, Miyara M, Moshous D, Magrin E, Denis A, Suarez F, Lagresle C, Roche AM, Everett J, Trinquand A, Guisset M, Bayford JX, Hacein-Bey-Abina S, Kauskot A, Elfeky R, Rivat C, Abbas S, Gaspar HB, Macintyre E, Picard C, Bushman FD, Galy A, Fischer A, Six E, Thrasher AJ, Cavazzana M. Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome. Nat Med. 2022 Jan;28(1):71-80. doi: 10.1038/s41591-021-01641-x. Epub 2022 Jan 24. Erratum in: Nat Med. 2022 Oct;28(10):2217. doi: 10.1038/s41591-022-01985-y. PMID: 35075289; PMCID: PMC8799465. Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome - PubMed

CD68, Cluster of Differentiation 68 Marker for monocyte lineage and circulating macrophages; ET3, FVIII variant with porcine A1 and ap-A3 domain sequences; HSCs, hematopoietic stem cells; LV, Lentivirus; VCN, vector copy number