Base editing corrects the common Salla disease SLC17A5 c.115C>T variant.

Free sialic acid storage disorders (FSASDs) result from pathogenic variations in the SLC17A5 gene, which encodes the lysosomal transmembrane protein sialin. Loss or deficiency of sialin impairs FSA transport out of the lysosome, leading to cellular dysfunction and neurological impairment, with the most severe form of FSASD resulting in death during early childhood. There are currently no therapies for FSASDs. Here, we evaluated the efficacy of CRISPR-Cas9-mediated homology directed repair (HDR) and adenine base editing (ABE) targeting the founder variant, SLC17A5 c.115C>T (p.Arg39Cys) in human dermal fibroblasts. We observed minimal correction of the pathogenic variant in HDR samples with a high frequency of undesired insertions/deletions (indels) and significant levels of correction for ABE-treated samples with no detectable indels, supporting previous work showing that CRISPR-Cas9-mediated ABE outperforms HDR. Furthermore, ABE treatment of either homozygous or compound heterozygous SLC17A5 c.115C>T human dermal fibroblasts demonstrated significant FSA reduction, supporting amelioration of disease pathology. Translation of this ABE strategy to mouse embryonic fibroblasts harboring the Slc17a5 c.115C>T variant in homozygosity recapitulated these results. Our study demonstrates the feasibility of base editing as a therapeutic approach for the FSASD variant SLC17A5 c.115C>T and highlights the usefulness of base editing in monogenic diseases where transmembrane protein function is impaired.

Neprilysin-2 is an important ß-amyloid degrading enzyme.

Proteases that degrade the amyloid-ß peptide (Aß) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aß accumulation (∼30- to 50-fold) and pathological deposition in rodents. The Aß-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (∼1.5- to 2-fold) in Aß, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aß. We investigated the role of NEP2 in Aß degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aß species in the hippocampus and brainstem/diencephalon (∼1.5-fold). Increases in Aß accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aß levels were marginally increased (∼1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aß, suggesting that yet other NEP-like Aß-degrading endopeptidases are contributing to Aß catabolism.

CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease.

Pompe disease, an autosomal recessive disorder caused by deficient lysosomal acid α-glucosidase (GAA), is characterized by accumulation of intra-lysosomal glycogen in skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant, the most frequent GAA pathogenic mutation in people of Southern Han Chinese ancestry, causes infantile-onset Pompe disease (IOPD), presenting neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, respiratory failure, and infantile mortality. We applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a Gaaem1935C>A knock-in mouse model and a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, histological, physiological, and behavioral characterization of 3-month-old homozygous Gaaem1935C>A mice. Homozygous Gaaem1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, had near-abolished GAA enzymatic activity, markedly increased tissue glycogen storage, and concomitantly impaired autophagy. Three-month-old mice demonstrated skeletal muscle weakness and hypertrophic cardiomyopathy but no premature mortality. The Gaaem1935C>A knock-in mouse model recapitulates multiple salient aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay.

PARP6, a member of a family of enzymes (17 in humans) known as poly-ADP-ribose polymerases (PARPs), is a neuronally enriched PARP. While previous studies from our group show that Parp6 is a regulator of dendrite morphogenesis in rat hippocampal neurons, its function in the nervous system in vivo is poorly understood. Here, we describe the generation of a Parp6 loss-of-function mouse model for examining the function of Parp6 during neurodevelopment in vivo. Using CRISPR-Cas9 mutagenesis, we generated a mouse line that expressed a Parp6 truncated variant (Parp6TR) in place of Parp6WT. Unlike Parp6WT, Parp6TR is devoid of catalytic activity. Homozygous Parp6TR do not exhibit obvious neuromorphological defects during development, but nevertheless die perinatally. This suggests that Parp6 catalytic activity is important for postnatal survival. We also report PARP6 mutations in six patients with several neurodevelopmental disorders, including microencephaly, intellectual disabilities, and epilepsy. The most severe mutation in PARP6 (C563R) results in the loss of catalytic activity. Expression of Parp6C563R in hippocampal neurons decreases dendrite morphogenesis. To gain further insight into PARP6 function in neurons we also performed a BioID proximity labeling experiment in hippocampal neurons and identified several microtubule-binding proteins (e.g., MAP-2) using proteomics. Taken together, our results suggest that PARP6 is an essential microtubule-regulatory gene in mice, and that the loss of PARP6 catalytic activity has detrimental effects on neuronal function in humans.

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness.

Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.

Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein.

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (V(H)) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of V(H) may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The V(H) sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies.

Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2.

B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5'-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

PARP6 is a Regulator of Hippocampal Dendritic Morphogenesis.

Mono-ADP-ribosylation (MARylation) of mammalian proteins was first described as a post-translational modification catalyzed by bacterial toxins. It is now known that endogenous MARylation occurs in mammalian cells and is catalyzed by 11 members of the poly-ADP-ribose polymerase (PARP) family of proteins (17 in humans). The physiological roles of these PARPs remain largely unknown. Here we demonstrate that PARP6, a neuronally enriched PARP that catalyzes MARylation, regulates hippocampal dendrite morphogenesis, a process that is critical for proper neural circuit formation during development. Knockdown of PARP6 significantly decreased dendritic complexity in embryonic rat hippocampal neurons in culture and in vivo. Expression of wild-type PARP6 increased dendritic complexity; conversely, expression of a catalytically inactive PARP6 mutant, or a cysteine-rich domain deletion mutant that has significantly reduced catalytic activity, decreased dendritic complexity. The identification of PARP6 as a regulator of dendrite morphogenesis supports a role for MARylation in neurons during development.

Altered NEP2 expression and activity in mild cognitive impairment and Alzheimer's disease.

Neprilysin-2 (NEP2), a close homolog of neprilysin (NEP), degrades amyloid-ß (Aß) and serves an important role in clearing Aß in vivo. We measured NEP2 and NEP mRNA levels from non-impaired (NI), mild cognitive impaired (MCI), and clinical Alzheimer's disease (AD) subjects in the mid-temporal gyrus, mid-frontal gyrus, caudate, and cerebellum. NEP2 activity levels were also determined. Our results indicate that NEP2 and NEP mRNA expression is altered in MCI subjects relative to NI subjects in AD-susceptible regions. NEP2 enzymatic activity was lowered in association with MCI and AD and was positively associated with cognitive function, independent of diagnostic category. Our finding that NEP2 expression and activity are altered in MCI is significant as these changes may potentially serve as preclinical markers for AD and reduced NEP2 activity may be associated with the development of AD.