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Pompe disease is a rare genetic neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency resulting in lysosomal glycogen accumulation and progressive myopathy. Enzyme replacement therapy, the current standard of care, penetrates poorly into the skeletal muscles and the peripheral and central nervous system (CNS), risks recombinant enzyme immunogenicity, and requires high doses and frequent infusions. Lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy was investigated in a Pompe mouse model using a clinically relevant promoter driving nine engineered GAA coding sequences incorporating distinct peptide tags and codon optimizations. Vectors solely including glycosylation-independent lysosomal targeting tags enhanced secretion and improved reduction of glycogen, myofiber, and CNS vacuolation in key tissues, although GAA enzyme activity and protein was consistently lower compared with native GAA. Genetically modified microglial cells in brains were detected at low levels but provided robust phenotypic correction. Furthermore, an amino acid substitution introduced in the tag reduced insulin receptor-mediated signaling with no evidence of an effect on blood glucose levels in Pompe mice. This study demonstrated the therapeutic potential of lentiviral HSPC gene therapy exploiting optimized GAA tagged coding sequences to reverse Pompe disease pathology in a preclinical mouse model, providing promising vector candidates for further investigation.

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNAmiR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34+ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNAmiR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNAmiR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNAmiR-containing vectors for RNAi-based therapy. Keywords: RNAi, CRISPR/Cas9, DROSHA microprocessor, recombinant viral vector titer, producer cell line