Hematopoietic and Immunological Assessment in Fanconi Anemia after Ex Vivo Lentiviral FANCA Gene Therapy with RP-L102

Introduction: Fanconi anemia (FA) is the most frequent cause of genetic bone marrow failure (BMF) resulting from inherited defects in the FA pathway, one of several essential deoxyribonucleic acid (DNA) repair mechanisms. Although allogeneic hematopoietic stem cell transplantation (alloHSCT) offers hematolymphoid cure, it confers significant morbidity and mortality and is thereby indicated only after development of severe BMF or dysplasia. Ex vivo hematopoietic stem and progenitor cell (HSPC)-based gene therapy (GT) offers an alternative strategy for earlier and potentially safer intervention with the goal of preventing BMF and the need for alloHSCT. Core assessments of safety and efficacy are conducted as part of ongoing GT clinical trials for FA-type A patients who have received ex vivo lentiviral FANCA GT. In these additional exploratory research studies we seek to better understand how GT may affect HSPC behavior, mutational acquisition and immune function.

Methods: To deepen our understanding of the effects of ex vivo lentiviral FANCA GT on FA biology, we collected serial bone marrow (BM) and peripheral blood (PB) samples from FA-Type A patients who were enrolled at Stanford University on the Phase I and II RP-L102-0418 and RP-L102-0319 clinical trials. We initiated the assessment of Phase I subjects at 18 months post treatment and the assessment of Phase II subjects at study enrollment. BM was assessed to characterize HSPC distribution by flow cytometry, proliferative capacity by colony-forming count (CFC) assays, transcriptomics by single-cell RNA sequencing (scRNASeq) and mutation acquisition by targeted next generation sequencing (NGS). Immune system composition and function were assessed using PB flow cytometry, immunoglobulin levels and T cell proliferative assays.

Results: Of the 8 enrolled patients, 5 subjects met eligibility criteria and were treated with RP-L102 GT. As of April 2022, 4 remain on study, now 18 to 36 months post-treatment. Of these 4 subjects, 3 have demonstrated increasing vector copy number and MMC-resistance. At time of enrollment, HSCs were markedly reduced in these patients compared to healthy controls, even in patients with no or mild cytopenia, and to-date have remained decreased post-GT. Functional HSPC proliferation capacity, as assessed by CFCs, was reduced in all patients at screening but showed evidence of improvement or stabilization in 3 of the 4 post-GT patients, as did BM total cellularity and CD34+ counts. scRNASeq showed reduced proportions of HSCs pre- and post-GT relative to healthy controls. Expression of ribosome biogenesis genes, an indicator of cancer risk, was downregulated post-GT. Targeted bulk-NGS revealed that none of the patients evaluated on these studies had mutations known to be associated with myeloid malignancies pre- or post-GT treatment. Detailed immune monitoring revealed that 3 patients had B-cell lymphopenia at time of enrollment. Additionally, 1 patient showed an increase in B-cell numbers post-GT. IgG levels were normal in all 8 screened patients pre-GT and remained stable post-GT. Interestingly, among 7 vaccinated patients evaluated at enrollment, 3 did not have protective IgG levels to Hepatitis B, 1 did not have protective IgG levels to Varicella Zoster Virus (VZV), and 3 showed equivocal levels of anti-VZV IgG.

Conclusions: Although our results are based on a small cohort and are interim within ongoing clinical trials, they indicate the need for more thorough assessment of BM and immune characterization in FA patients beyond reliance on blood counts. Importantly, the paucity of HSCs in FA patients even prior to the development of cytopenias supports the need for early intervention for HSC preservation which may be possible with GT upon diagnosis. Additionally, these studies show that RP-L102 GT appears to be a safe approach for hematopoietic preservation in FA without acquisition of detectable mutations in HSPCs to-date. Moreover, downregulation of ribosome biogenesis genes can potentially lead to decreased leukemogenesis which could decrease cancer risk post-GT. Finally, we show laboratory evidence of baseline immune dysfunction in FA patients mainly consisting of B-cell lymphopenia and poor response to certain vaccines. Given this, post-GT re-vaccination of patients may be clinically indicated and would enable assessment of functional immune recovery potentially associated with the GT treatment.

Felber:Jazz Pharmaceuticals: Research Funding. Choi:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Czechowicz:STRM.BIO: Research Funding; Rocket Pharmaceuticals, Inc.: Research Funding; Beam Therapeutics: Consultancy, Current equity holder in publicly-traded company; GV: Consultancy, Current equity holder in publicly-traded company; Spotlight Therapeutics: Consultancy, Current equity holder in publicly-traded company; Stemodontics: Consultancy, Current holder of stock options in a privately-held company; Global Blood Therapeutics: Current equity holder in publicly-traded company; Editas Medicine: Current equity holder in publicly-traded company; Magenta Therapeutics: Current equity holder in publicly-traded company, Other: Intellectual Property Rights; Decibel Therapeutics: Current equity holder in publicly-traded company; Gilead Sciences: Other: Intellectual Property Rights; Jasper Therapeutics: Other: Intellectual Property Rights; Teiko Bio: Consultancy, Current holder of stock options in a privately-held company.