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Two unique gene mutations in the enzyme beta-glucuronidase (GUSB) that result in the lysosomal storage disease Mucopolysaccharidosis (MPS) type VII had previously been reported to have differing disease phenotype severities when compared on differing mouse strains. The MPSVII mouse has proven to be a highly efficacious model to study mucopolysaccharidoses and for evaluating potential gene or stem cell therapies for lysosomal storage diseases. We examined the single base pair deletion (MPSVII) and the intracisternal A particle element insertion (MPSVII2J) in GUSB compared with control animals by skeletal measures, electroretinography, auditory-evoked brainstem response and life span on a C57BL/6J background strain. In all measures, both mutations result in either a trend toward or significant changes from the background strain control. In all measures, there is no significant phenotypic difference between the two mutations. The 2J variant is a more easily genotyped and equally affected phenotype, which holds promise for further studies of chimerism and stem cell therapy approaches., (© 2021 The Authors.)

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.

Jak2 is a member of the Janus family of tyrosine kinases and is involved in cytokine signaling. As a part of a study to determine biological functions of Jak2, we used molecular modeling to identify W1038 as a residue that is critical for tyrosine kinase function. Mutation of W1038, in tandem with E1046, generates a dominant-negative form of the Jak2 protein. Mice that were engineered to express two copies of this dominant-negative Jak2 protein died in utero. Additionally, heterozygous mice expressing Jak2 with kinase activity that is moderately reduced when compared to wild-type activity appear phenotypically normal. Collectively, these data suggest that Jak2 kinase activity is essential for normal mammalian development.

Non-viral gene transfer is an alternative to viral vectors for gene transfer. However, non-viral transgene expression remains undesirably low and transient. Matrix attachment regions (MARs) are DNA elements that are defined by their high affinity for the nuclear matrix. MARs may also be related to long-term transgene expression in vitro. The purpose of this research is to evaluate human interferon-beta MARs element in various cell types. This was accomplished by constructing MARs-containing plasmid DNA (pDNA) and comparing their transgene expression with non-MARs-containing pDNA. We found that MARs-containing pDNA increased and prolonged the expression in Chinese hamster ovary (CHO) cells, but not in human neuroblastoma cells (SKnSH) and neuronal cells (primary neuron, astroglia and microglia). From the cotransfection experiment, MARs-containing pDNA had a trans effect on another pDNA expression. A PCR method was used to monitor the intracellular distribution of pDNA after cellular fractionalization. We found that non-MAR containing pDNA demonstrates similar intracellular distribution as non-MARs-containing pDNA.