<i>In Vivo</i> Hematopoietic Stem Cell Engineering Restores the Function of NADPH Enzyme Complex in X-Linked Chronic Granulomatous Disease Model Mice

X-linked chronic granulomatous disease (X-CGD) is a rare primary immune deficiency disorder caused by a defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex, which impairs the ability of phagocytic cells to eliminate bacterial and fungal pathogens with reactive oxygen species. Allogeneic hematopoietic stem cell (HSC) transplant is the standard treatment for X-CGD patients but requires a suitable donor and carries the risk of graft rejection, graft-vs-host disease, and other co-morbidities. Chronic antibacterial and antifungal prophylaxis are also available, but many patients still experience bouts of severe infection and chronic inflammatory complications. Although ex vivo autologous HSC gene therapy demonstrated clinical safety and efficacy in X-CGD patients, it carries the risks and burden of conditioning and is manufactured on a per-patient basis, which limits access. To overcome the limitations in current treatments and address an unmet medical need, Ensoma developed a novel in vivo gene therapy for X-CGD that uses virus-like particles (VLP) based on helper-dependent adenovirus 3/35++ for in vivo transduction and engineering of HSCs. Our drug product, EN-374, consists of two independent VLPs. One VLP contains DNA encoding Sleeping Beauty 100X transposase and Flp recombinase. The other VLP contains a transposon with a myeloid specific promoter expressing wild-type CYBB sequence and a ubiquitous promoter expressing a selectable methylguanine methyltransferase (MGMT) marker containing the P140K variant that prevents binding to O6-benzylguanine (O6BG), an inhibitor of MGMT activity. In cells co-transduced by the two VLPs, the transposon is excised and integrated into the genomic DNA to drive stable and sustained expression of CYBB and restore NADPH oxidase activity in neutrophils. During the enrichment phase, unmodified HSCs are depleted by O6BG/TMZ while gene-modified HSCs expressing MGMT-P140K are protected from the toxic effects of TMZ and allowed to expand to achieve therapeutic levels of gene marking. EN-374 targets the receptor CD46 on HSCs for entry into cells, therefore a transgenic mouse line (CD46tg) was utilized that expresses human CD46 on mouse cells comparable to human CD46 tissue expression pattern and protein expression levels. The CD46tg mice were crossed with CYBB knockout (KO) mice (The Jackson Laboratory B6.129S-Cybbtm1Din/J) to generate a disease relevant animal model which mimics the lack of NADPH oxidative activity in human X-CGD patients (CD46tg/CYBB KO). In an in vivo study, CD46tg/CYBB KO mouse HSCs were first mobilized from the bone marrow to peripheral circulation by granulocyte colony stimulating factor and plerixafor. VLP drug product was administered at the peak of HSC mobilization. Gene-modified HSCs in the peripheral circulation homed back to the bone marrow and, via the selectable MGMT-P140K marker, were enriched by administration of O6BG and temozolomide. Post enrichment, integration analysis demonstrated payload vector copy number of EN-374 treated mice was 0.51 ± 0.14 vector copy number/cell in bulk bone marrow. To determine restored CYBB protein expression and NADPH activity in vivo, circulating neutrophils (CD11b+/Ly6G+) were evaluated throughout the study by a flow cytometry-based dihydrorhodamine (DHR) assay. At the end of study, in the EN-374-treated group, the average CYBB protein expression and DHR activity in circulating neutrophils was 30.7 ± 9.9 % and 41.0 ± 11.7 % (mean ± SEM), respectively. Restoring ≥10% of neutrophils with NADPH oxidase activity has been shown to confer clinically meaningful improvements in infection outcomes in patients with X-CGD. Our data demonstrate compelling proof-of-concept for efficient in vivo engineering of HSCs that leads to the expression of functional CYBB at therapeutic levels in X-CGD disease model mice. This study sets the foundation for the first in vivo HSC gene therapy to reach the clinic and can be applied to a range of genetic disorders which thus far have only been addressed with ex vivo gene therapies that involve significant patient burden and manufacturing limitations. Thus, this VLP-based, off-the-shelf therapy has the potential to dramatically broaden the patient populations that can benefit from HSC-directed gene therapies.