Comparative dose effectiveness of intravenous and intrathecal AAV9.CB7.hIDS, RGX-121, in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disease caused by iduronate-2-sulfatase (IDS) deficiency, leading to accumulation of glycosaminoglycans (GAGs) and the emergence of progressive disease. Enzyme replacement therapy is the only currently approved treatment, but it leaves neurological disease unaddressed. Cerebrospinal fluid (CSF)-directed administration of AAV9.CB7.hIDS (RGX-121) is an alternative treatment strategy, but it is unknown if this approach will affect both neurologic and systemic manifestations. We compared the effectiveness of intrathecal (i.t.) and intravenous (i.v.) routes of administration (ROAs) at a range of vector doses in a mouse model of MPS II. While lower doses were completely ineffective, a total dose of 1 × 109 gc resulted in appreciable IDS activity levels in plasma but not tissues. Total doses of 1 × 1010 and 1 × 1011 gc by either ROA resulted in supraphysiological plasma IDS activity, substantial IDS activity levels and GAG reduction in nearly all tissues, and normalized zygomatic arch diameter. In the brain, a dose of 1 × 1011 gc i.t. achieved the highest IDS activity levels and the greatest reduction in GAG content, and it prevented neurocognitive deficiency. We conclude that a dose of 1 × 1010 gc normalized metabolic and skeletal outcomes, while neurologic improvement required a dose of 1 × 1011 gc, thereby suggesting the prospect of a similar direct benefit in humans.

Generation and characterization of an immunodeficient mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II) is an inherited X-linked recessive disease caused by deficiency of iduronate-2-sulfatase (IDS), resulting in the accumulation of the glycosaminoglycans (GAG) heparan and dermatan sulfates. Mouse models of MPS II have been used in several reports to study disease pathology and to conduct preclinical studies for current and next generation therapies. Here, we report the generation and characterization of an immunodeficient mouse model of MPS II, where CRISPR/Cas9 was employed to knock out a portion of the murine IDS gene on the NOD/SCID/Il2rγ (NSG) immunodeficient background. IDS-/- NSG mice lacked detectable IDS activity in plasma and all analyzed tissues and exhibited elevated levels of GAGs in those same tissues and in the urine. Histopathology revealed vacuolized cells in both the periphery and CNS of NSG-MPS II mice. This model recapitulates skeletal disease manifestations, such as increased zygomatic arch diameter and decreased femur length. Neurocognitive deficits in spatial memory and learning were also observed in the NSG-MPS II model. We anticipate that this new immunodeficient model will be appropriate for preclinical studies involving xenotransplantation of human cell products intended for the treatment of MPS II.

Phenotypic Correction of Murine Mucopolysaccharidosis Type II by Engraftment of Ex Vivo Lentiviral Vector-Transduced Hematopoietic Stem and Progenitor Cells.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal disease caused by deficiency of iduronate-2-sulfatase (IDS). The absence of IDS results in the accumulation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Currently, the only approved treatment option for MPS II is enzyme replacement therapy (ERT), Elaprase. However, ERT is demanding for the patient and does not ameliorate neurological manifestations of the disease. Using an IDS-deficient mouse model that phenocopies the human disease, we evaluated hematopoietic stem and progenitor cells (HSPCs) transduced with a lentiviral vector (LVV) carrying a codon-optimized human IDS coding sequence regulated by a ubiquitous MNDU3 promoter (MNDU3-IDS). Mice treated with MNDU3-IDS LVV-transduced cells showed supraphysiological levels of IDS enzyme activity in plasma, peripheral blood mononuclear cells, and in most analyzed tissues. These enzyme levels were sufficient to normalize GAG storage in analyzed tissues. Importantly, IDS levels in the brains of MNDU3-IDS-engrafted animals were restored to 10-20% than that of wild-type mice, sufficient to normalize GAG content and prevent emergence of cognitive deficit as evaluated by neurobehavioral testing. These results demonstrate the potential effectiveness of ex vivo MNDU3-IDS LVV-transduced HSPCs for treatment of MPS II.

Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency.

During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T-B-NK+ severe combined immunodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning regimens are poorly tolerated, leading to early mortality and/or late complications, including short stature, endocrinopathies, and dental aplasia. However, without alkylating chemotherapy as preconditioning, patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We designed a self-inactivating lentivirus vector containing human Artemis cDNA under transcriptional regulation of the human endogenous Artemis promoter (AProArt). Fibroblasts from ART-SCID patients transduced with AProArt lentivirus showed correction of radiosensitivity. Mobilized peripheral blood CD34+ cells from an ART-SCID patient as well as hematopoietic stem cells from Artemis-deficient mice demonstrated restored T and B cell development following AProArt transduction. Murine hematopoietic cells transduced with AProArt exhibited no increase in replating potential in an in vitro immortalization assay, and analysis of AProArt lentivirus insertions showed no predilection for sites that could activate oncogenes. These efficacy and safety findings support institution of a clinical trial of gene addition therapy for ART-SCID.

Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID.

Characterization of the human artemis promoter by heterologous gene expression in vitro and in vivo.

Artemis is an endonucleolytic enzyme involved in nonhomologous double-strand break repair and V(D)J recombination. Deficiency of Artemis results in a B- T- radiosensitive severe combined immunodeficiency, which may potentially be treatable by Artemis gene transfer into hematopoietic stem cells. However, we recently found that overexpression of Artemis after lentiviral transduction resulted in global DNA damage and increased apoptosis. These results imply the necessity of effecting natural levels of Artemis expression, so we isolated a 1 kilobase DNA sequence upstream of the human Artemis gene to recover and characterize the Artemis promoter (APro). The sequence includes numerous potential transcription factor-binding sites, and several transcriptional start sites were mapped by 5' rapid amplification of cDNA ends. APro and deletion constructs conferred significant reporter gene expression in vitro that was markedly reduced in comparison to expression regulated by the human elongation factor 1-α promoter. Ex vivo lentiviral transduction of an APro-regulated green fluorescent protein (GFP) construct in mouse marrow supported GFP expression throughout hematopoeitic lineages in primary transplant recipients and was sustained in secondary recipients. The human Artemis promoter thus provides sustained and moderate levels of gene expression that will be of significant utility for therapeutic gene transfer into hematopoeitic stem cells.

Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase.

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.