Gene Therapy for Inherited Blood Disorders

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Objective 1: Develop a lentiviral viral (LV) and foamy viral (FV) vector-based gene therapy approach for the treatment of subjects with LAD-1 1.1 Pre-clinical studies to evaluate the efficacy of LV in human LAD-1 CD34+ cells We are conducting pre-clinical studies to investigate the efficacy of LV bearing an MND-promoter-driven CD18 expression cassette (LV-CD18) to mediate CD18 expression and cellular phenotypic correction in transduced human LAD-1 HSPCs. Human LAD-1 CD34+ cells were transduced ex vivo with LV-hCD18 with or without P407 and PGE2. At six months following transplantation of LV-hCD18-transduced LAD-1 HSPCs into immuno-deficient (NSG) mice, animals that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 0.40% CD18+ human myeloid cells, whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE2 showed 9.56 0.96% CD18+ human myeloid cells, thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. This work was submitted to the 2021 ASH Annual Meeting. 1.2 Pre-clinical studies to evaluate the efficacy of FV in human LAD-1 CD34+ cells We have conducted extensive pre-clinical studies to investigate the efficacy of clinical grade FV expressing the human CD18 cDNA (FV-hCD18) in HSPCs collected from subjects with LAD-1. Cells were transduced ex vivo with FV-hCD18 for 16 hours and CD18+ cell surface expression was demonstrated in 39-42% of cells. Genetic correction of HSPCs from LAD-1 patients restored the chemotactic function of neutrophils differentiated from these progenitor cells in vitro. Transplantation of FV-hCD18-transduced LAD-1 HSPCs into NSG mice resulted in 36.0 3.9% of human cells expressing CD18 in the murine BM five months after transplantation. A polyclonal pattern of integration was observed with no evidence of insertional mutagenesis five months after transplantation. A first-in-human phase I/II gene therapy clinical trial using FV for the gene therapy of LAD-1 was designed but accrual is on hold due to delays in production of GMP grade FV. This work is under peer-review Molecular Therapy (2021). Objective 2: Develop and evaluate safety of CRISPR/Cas9-based strategies for genome editing in human HSPCs 2.1 Homology-independent targeted integration (HITI) In this study, we utilized a homology-independent targeted integration (HITI)-based approach to achieve robust site-specific transgene integration in human adult CD34+ HSPCs. As proof-of-concept, a reporter gene was targeted to a clinically relevant genetic locus (ITGB2) using a rAAV6 vector and sgRNA/Cas9 RNP complexes. We demonstrate high levels of stable HITI-mediated genome editing (21%) in repopulating HSPCs after transplant into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs for the treatment of monogenic diseases affecting the hematopoietic system. This work was published Molecular Therapy 29 (4): 1611-1624 (2021), and J. Clin. Med. 10 (3); 513 (2021). 2.2 Enhancing HDR-mediated gene editing approaches with baculovirus vectors One of the major hurdles of HDR-mediated gene editing approaches is viral delivery of large DNA templates needed for gene correction. In this study, we investigated baculovirus (BV), a large dsDNA virus with virtually unlimited packaging capacity, as an alternative delivery approach for gene editing. We transduced human CD34+ cells with VSV-G pseudotyped BV vectors harboring a copGFP reporter flanked by 4kb homology arms (HAs) to ITGB2, a locus mutated in LAD-1. In initial experiments, GFP expression in transduced CD34+CD38+ progenitors and CD34+CD38- HSC-enriched populations was largely undetectable. Based on recent publications indicating potent BV-mediated activation of cellular death and innate immune pathways, including TLRs and cGAS-STING, we systematically tested the impact of small molecule inhibitors targeting apoptosis, necroptosis, pyroptosis and major dsDNA sensing innate immune on BV transduction. We identified a unique combination of three inhibitors, H-151, z-VAD-FMK and Nec-1. Repeat transduction in the presence of this optimized drug cocktail led to marked improvement in GFP expression after BV transduction, with mean GFP+ percentages of 28% and 8% in CD34+CD38- and CD34+CD38+ cells, respectively. Collectively, our results lay the groundwork for future studies characterizing innate immune responses to dsDNA viruses in CD34+ cells, and highlight the potential use of BV as a delivery system for HDR-mediated gene editing in HSPCs. This work was submitted to the 2021 ASH Annual Meeting. 2.3 Genome-wide analysis of off-target CRISPR/Cas9 activity in gene-edited human HSPCs We electroporated Cas9 ribonucleoprotein (RNP) complexes into human adult HSPCs and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via high-throughput, whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified over 21,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel indels among the samples. The average number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but lower than the average number observed in an electroporation-only control group. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We conclude that the collective somatic mutational burden associated with genome editing in human HSPCs utilizing Cas9 RNP complexes targeted to at least two different chromosomal loci is indistinguishable from naturally occurring levels of background genetic heterogeneity. This work was published Genes 11 (12): 1501 (2020). 2.4 Impact of G-CSF use after transplantation of gene-edited HSPCs We subcutaneously injected G-CSF or a vehicle control solution from day 1 to 14 following transplantation of NSG mice with human CD34+ cells edited using CRISPR/Cas9 approaches at the AAVS1 locus. G-CSF treated mice had significantly lower PB, BM and splenic engraftment at the endpoint (22 weeks) analysis. Percentages of immunophenotypic HSC subpopulations were 2-fold lower within the BM of G-CSF treated mice. In serial limiting dilution analyses in secondary mice, we measured a 5.1-fold reduced frequency of LTR-HSCs in mice injected with G-CSF compared with the untreated group (p = 0.011). Collectively, our data suggest that G-CSF use post-transplant significantly reduces LTR and self-renewal capacities of CRISPR-Cas9 gene edited HSPCs. Objective 3: Develop a targeted, non-genotoxic conditioning regimen based on anti-c-MPL antibodies conjugated to immunotoxin. Construction of anti-c-MPL bivalent single-chain fragment variable (biscFV) fused with the diphtheria toxin residues 1-390 (DT390) was completed in FY21. The resulting DT390-biscFV(c-MPL) (96.5 KDa) was produced using the yeast Pichia pastoris expression system. Proof-of-concept experiments using HEK293A, and human or NHP CD34+ cells showed marked cMPL-dependent cytotoxic effect in vitro. Similarly, DT390-biscFV (cMPL) could safely target and deplete human HSPCs in vivo in humanized NBSGW immunodeficient mice. Notably, after a single i.v. administration (0.3mg/kg) in a rhesus macaque model, we observed >80% depletion of the most primitive CD34+CD38-CD45RA-CD90+CD49f+ cells within the marrow. Half-life was 38 minutes. Transplantation of gene marked CD34+ cells following DT390-biscFV(cMPL) conditioning of additional NHPs will be performed in FY22.