Gene Therapy for Inherited Blood Disorders

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Objective 1: Develop novel HSPC gene correction strategies Because multiple causative mutations have been identified in FA, targeted introduction of a therapeutic open reading frame at the endogenous FANCA genetic locus is desirable to allow single-construct genetic correction regardless of the nature of downstream mutations. Due to faulty DNA repair mechanisms in FA cells, genome editing approaches that rely on CRISPR-Cas9-mediated DNA DSBs and subsequent repair are impractical in FA HSPCs. Accordingly, we are developing novel strategies that bypass the DNA damage response (DDR) for targeted integration. To this end, we have incorporated the programmability of CRISPR-Cas9 with the integration efficiency of DNA recombinase domains known to mediate enzymatic DNA ligation reactions without formation of de novo DNA free ends. Namely, we have constructed chimeric proteins with nuclease-deficient mutant of Cas9 (dCas9) molecularly fused to tandem domains of known eukaryotic transposases (e.g., Tn5, PiggyBac, and Sleeping Beauty) or HIV integrase to enable dCas9-directed, recombinase-mediated, DDR-independent integration of an FA donor substrate at the FANCA genetic locus. Testing of these approaches is underway. Objective 2: Facilitate safe and efficient engraftment of gene-edited HSPCs Pre-transplant conditioning with chemotherapy-free regimens. We hypothesized that cMPL might be a relevant antigen for an antibody-based targeted depletion of human HSPCs and provide the basis for a safer conditioning regimen prior to transplant. To investigate this possibility, we have produced a recombinant anti-cMPL bivalent (bi) single-chain fragment variable (scFV) fused with diphtheria toxin (DT) truncated at residue 390 (DT390-biscFV(cMPL)). This agent has enabled HSPC depletion in pre-clinical in vitro and non-human primate (NHP) models. Further optimization of dosage is underway in a knock-in mouse model harboring human TPO and cMPL gene sequences. We are also pursuing autologous transplantation of genetically barcoded HSPCs in NHPs conditioned with DT390-biscFV(cMPL). The safety and efficacy profiles is monitored long-term and detailed quantitative longitudinal follow-up is performed in barcoded animals to assess stability of contributions from engrafted HSPC clones. Increase cell dose by ex vivo expansion of gene-edited HSPCs. To develop a clinically feasible platform for the expansion of genetically modified long-term repopulating adult HSPCs, we are building upon recent advances to develop a synthetic, cytokine-free expansion culture system that addresses the limited efficacy and batch-to-batch variability of current approaches. Three strategies are combined to further optimize culture conditions for HSPC expansion: 1) Promoting HSPC self-renewal. Key transcriptional regulators (e.g., HOXB4) have been identified as potential targets to enhance HSPC self-renewal in culture. Because constitutive expression of growth-promoting transcription factors (TFs) by viral transduction poses safety risks, we transiently express single or combined self-renewal regulators within target HSPCs in culture using lipid nanoparticle (LNP)-based transfer of TF mRNA; 2) Suppressing HSPC differentiation. We have recently identified 78c, a potent inhibitor of the CD38 differentiation marker, as a lead synthetic candidate for active suppression of HSPC differentiation in culture. 3) Mitigating endoplasmic reticulum (ER) stress. Recent studies have highlighted how increased proliferative demand triggers ER stress perturbations that collectively impair HSC function in ex vivo culture. To limit activation of ER stress pathways during expansion, we evaluate HSPC cultures under hypoxic conditions and supplemented with synthetic agonists of Hsf1 (e.g., 17-AAG) recently shown to limit ER stress by rebalancing proteostasis. Increase cell fitness by overcoming innate immune responses. A growing body of experimental evidence suggests a pivotal role of host antiviral factors and nucleic acid sensors in limiting the efficacy of HSPC genetic manipulation. To characterize innate immune pathways triggered by reagents used for genetic engineering of HSPCs, we are conducting unbiased proteomic and single-cell transcriptomic screens on human HSC-enriched populations exposed to commonly used gene delivery systems, including vectors based on RV, LV, FV and AAV6, as well electroporation and lipid nanoparticle carriers of nucleic acid constituents (e.g., DNA and mRNA). These findings will inform novel approaches to overcome immune blocks to nucleic acid delivery and enhance gene correction efficiencies by promoting cellular survival and fitness. Evaluate the impact of post-transplant G-CSF administration on gene-edited HSPCs. Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. We provided evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA DSBs. Transient p53 inhibition in culture attenuated the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF did not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. BioRxiv 2023: doi: 10.1101/2023.06.29.547089. Objective 3: Develop in vivo gene therapy strategies In vivo delivery of genetic payloads could circumvent the shortcomings of current ex vivo gene correction approaches and represent a distinct advance for gene therapy of Fanconi anemia. Among available in vivo delivery methods, LNPs are the most developed for clinical use. A 3-step preclinical study is underway to provide a comprehensive evaluation of the efficiency (Aim 1), safety (Aim 2) and therapeutic applicability (Aim 3) of novel LNP delivery systems. Aim 1- In pilot experiments, we have identified and optimized a novel LNP formulation based on the ionizable dendrimer amino lipid 4A3-SC852, and shown efficacy for delivery of nucleic acid cargoes to purified human HSPCs in vitro. Building on our previous studies demonstrating cMPL as a relevant antigen to target HSPCs, optimized LNP formulations have been chemically conjugated to a recombinant anti-cMPL bivalent single-chain fragment variable (biscFV(cMPL)) to facilitate selective delivery of CRISPR reagents to HSPCs in a NHP model in vivo. Aim 2- Specificity of LNP-mediated delivery of CRISPR reagents to HSPCs is imperative to limit off-target gene editing and systemic toxicity in vivo. To address this question, biodistribution of LNP formulations administered intravenously to NHPs are investigated using established in vivo tracking approaches. Briefly, biscFV(cMPL)-conjugated LNP formulations are encapsulated with the sodium/iodide symporter (NIS) mRNA, infused to the animals, and tracked by whole-body PET/CT scan imaging at select timepoints following intravenous injection of an 18F-tetrafluoroborate radiotracer. Aim 3- Integration of an FA donor substrate at the endogenous locus within HSPCs will be pursued in NHPs by cMPL-conjugated LNP delivery of a dCas9-directed, recombinase-mediated, DDR-independent genome editing system.