Improved Gene Therapy for Metachromatic Leukodystrophy

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed. The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021. We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. Disclosures Vanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Double paternal uniparental isodisomy 7 and 15 presenting with Beckwith-Wiedemann spectrum features

Here we describe for the first time double paternal uniparental isodisomy (iUPD) 7 and 15 in a baby boy with features in the Beckwith-Wiedemann syndrome spectrum (BWSp) (placentomegaly, hyperinsulinism, enlarged viscera, hemangiomas, and earlobe creases) in addition to conjugated hyperbilirubinemia. His phenotype was also reminiscent of genome-wide paternal uniparental isodisomy. We discuss the most likely origin of the UPDs; a maternal double monosomy 7 and 15 rescued by duplication of the paternal chromosomes after fertilization. So far, paternal UPD7 is not associated with an abnormal phenotype, while paternal UPD15 causes Angelman syndrome. Methylation analysis for other clinically relevant imprinting disorders, including BWSp, was normal. Therefore, we hypothesized that the double UPD affected other imprinted genes. To look for such effects, patient fibroblast RNA was isolated and analyzed for differential expression compared to six controls. We did not find apparent transcription differences in imprinted genes outside chromosomes 7 and 15 in patient fibroblast. PEG10 (7q21.3) was the only paternally imprinted gene on these chromosomes upregulated beyond double-dose expectation (6-fold). We speculate that a high PEG10 level could have a growth-promoting effect as his phenotype was not related to aberrations in BWS-locus on 11p15.5 after DNA, RNA, and methylation testing. However, many genes in gene sets associated with growth were upregulated. This case broadens the phenotypic spectrum of UPDs but did not show evidence of involvement of an imprinted gene network.

SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency

Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.