Haploinsufficiency underlies the neurodevelopmental consequences of SLC6A1 variants.

Heterozygous variants in SLC6A1, encoding the GAT-1 GABA transporter, are associated with seizures, developmental delay, and autism. The majority of affected individuals carry missense variants, many of which are recurrent germline de novo mutations, raising the possibility of gain-of-function or dominant-negative effects. To understand the functional consequences, we performed an in vitro GABA uptake assay for 213 unique variants, including 24 control variants. De novo variants consistently resulted in a decrease in GABA uptake, in keeping with haploinsufficiency underlying all neurodevelopmental phenotypes. Where present, ClinVar pathogenicity reports correlated well with GABA uptake data; the functional data can inform future reports for the remaining 72% of unscored variants. Surface localization was assessed for 86 variants; two-thirds of loss-of-function missense variants prevented GAT-1 from being present on the membrane while GAT-1 was on the surface but with reduced activity for the remaining third. Surprisingly, recurrent de novo missense variants showed moderate loss-of-function effects that reduced GABA uptake with no evidence for dominant-negative or gain-of-function effects. Using linear regression across multiple missense severity scores to extrapolate the functional data to all potential SLC6A1 missense variants, we observe an abundance of GAT-1 residues that are sensitive to substitution. The extent of this missense vulnerability accounts for the clinically observed missense enrichment; overlap with hypermutable CpG sites accounts for the recurrent missense variants. Strategies to increase the expression of the wild-type SLC6A1 allele are likely to be beneficial across neurodevelopmental disorders, though the developmental stage and extent of required rescue remain unknown.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

Predicting disease severity in metachromatic leukodystrophy using protein activity and a patient phenotype matrix.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene (ARSA) and categorized into three subtypes according to age of onset. The functional effect of most ARSA mutants remains unknown; better understanding of the genotype-phenotype relationship is required to support newborn screening (NBS) and guide treatment. RESULTS: We collected a patient data set from the literature that relates disease severity to ARSA genotype in 489 individuals with MLD. Patient-based data were used to develop a phenotype matrix that predicts MLD phenotype given ARSA alleles in a patient's genotype with 76% accuracy. We then employed a high-throughput enzyme activity assay using mass spectrometry to explore the function of ARSA variants from the curated patient data set and the Genome Aggregation Database (gnomAD). We observed evidence that 36% of variants of unknown significance (VUS) in ARSA may be pathogenic. By classifying functional effects for 251 VUS from gnomAD, we reduced the incidence of genotypes of unknown significance (GUS) by over 98.5% in the overall population. CONCLUSIONS: These results provide an additional tool for clinicians to anticipate the disease course in MLD patients, identifying individuals at high risk of severe disease to support treatment access. Our results suggest that more than 1 in 3 VUS in ARSA may be pathogenic. We show that combining genetic and biochemical information increases diagnostic yield. Our strategy may apply to other recessive diseases, providing a tool to address the challenge of interpreting VUS within genotype-phenotype relationships and NBS.

Identifying therapeutic drug targets using bidirectional effect genes.

Prioritizing genes for translation to therapeutics for common diseases has been challenging. Here, we propose an approach to identify drug targets with high probability of success by focusing on genes with both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). We find 98 BEST genes for a variety of indications. Drugs targeting those genes are 3.8-fold more likely to be approved than non-BEST genes. We focus on five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants in those genes result in significantly increased risk for idiopathic short stature (ISS) (OR = 2.75, p = 3.99 × 10-8). Finally, using functional experiments, we demonstrate that adding an exogenous CNP analog (encoded by NPPC) rescues the phenotype, thus validating its potential as a therapeutic treatment for ISS. Our results show the value of looking for bidirectional effects to identify and validate drug targets.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Virus-receptor mediated transduction of dendritic cells by lentiviruses enveloped with glycoproteins derived from Semliki Forest virus.

Lentiviruses have recently attracted considerable interest for their potential as a genetic modification tool for dendritic cells (DCs). In this study, we explore the ability of lentiviruses enveloped with alphaviral envelope glycoproteins derived from Semliki Forest virus (SFV) to mediate transduction of DCs. We found that SFV glycoprotein (SFV-G)-pseudotyped lentiviruses use C-type lectins (DC-SIGN and L-SIGN) as attachment factors for transduction of DCs. Importantly, SFV-G pseudotypes appear to have enhanced transduction towards C-type lectin-expressing cells when produced under conditions limiting glycosylation to simple high-mannose, N-linked glycans. These results, in addition to the natural DC tropism of SFV-G, offer evidence to support the use of SFV-G-bearing lentiviruses to genetically modify DCs for the study of DC biology and DC-based immunotherapy.

Pseudotyping lentiviral vectors with aura virus envelope glycoproteins for DC-SIGN-mediated transduction of dendritic cells.

Lentiviral vectors (LVs) pseudotyped with envelope proteins of alphaviruses have recently attracted considerable interest for their potential as gene delivery tools. We report the production of human immunodeficiency virus type 1 (HIV-1)-derived LVs pseudotyped with envelope glycoproteins derived from the Aura virus (AURA). We found that the AURA-glycoprotein-pseudotyped LVs use C-type lectins (DC-SIGN and L-SIGN) as attachment factors. These interactions with DC-SIGN are specific as determined by inhibition assays and appear to facilitate transduction through a pH-dependent pathway. AURA-pseudotyped LVs were used to transduce monocyte-derived dendritic cells (DCs) and the transduction was shown to be DC-SIGN mediated, as illustrated by competitive inhibition with DC-SIGN and L-SIGN antibodies and yeast mannan. Comparisons with LVs enveloped with glycoproteins derived from vesicular stomatitis virus and Sindbis virus suggest that AURA-glycoprotein-bearing LVs might be useful to genetically modify DCs for the study of DC biology and DC-based immunotherapy.

Production of lentiviral vectors with enhanced efficiency to target dendritic cells by attenuating mannosidase activity of mammalian cells.

BACKGROUND: Dendritic cells (DCs) are antigen-presenting immune cells that interact with T cells and have been widely studied for vaccine applications. To achieve this, DCs can be manipulated by lentiviral vectors (LVs) to express antigens to stimulate the desired antigen-specific T cell response, which gives this approach great potential to fight diseases such as cancers, HIV, and autoimmune diseases. Previously we showed that LVs enveloped with an engineered Sindbis virus glycoprotein (SVGmu) could target DCs through a specific interaction with DC-SIGN, a surface molecule predominantly expressed by DCs. We hypothesized that SVGmu interacts with DC-SIGN in a mannose-dependent manner, and that an increase in high-mannose structures on the glycoprotein surface could result in higher targeting efficiencies of LVs towards DCs. It is known that 1-deoxymannojirimycin (DMJ) can inhibit mannosidase, which is an enzyme that removes high-mannose structures during the glycosylation process. Thus, we investigated the possibility of generating LVs with enhanced capability to modify DCs by supplying DMJ during vector production. RESULTS: Through western blot analysis and binding tests, we were able to infer that binding of SVGmu to DC-SIGN is directly related to amount of high-mannose structures on SVGmu. We also found that the titer for the LV (FUGW/SVGmu) produced with DMJ against 293T.DCSIGN, a human cell line expressing the human DC-SIGN atnibody, was over four times higher than that of vector produced without DMJ. In addition, transduction of a human DC cell line, MUTZ-3, yielded a higher transduction efficiency for the LV produced with DMJ. CONCLUSION: We conclude that LVs produced under conditions with inhibited mannosidase activity can effectively modify cells displaying the DC-specific marker DC-SIGN. This study offers evidence to support the utilization of DMJ in producing LVs that are enhanced carriers for the development of DC-directed vaccines.

Lentiviral vectors for immune cells targeting.

Lentiviral vectors (LVs) are efficient gene delivery vehicles suitable for delivering long-term transgene expression in various cell types. Engineering LVs to have the capacity to transduce specific cell types is of great interest to advance the translation of LVs toward the clinic. Here we provide an overview of innovative approaches to target LVs to cells of the immune system. In this overview we distinguish between two types of LV targeting strategies: (i) targeting of the vectors to specific cells by LV surface modifications, and (ii) targeting at the level of transgene transcription by insertion of tissue-specific promoters to drive transgene expression. It is clear that each strategy is of enormous value but ultimately combining these approaches may help reduce the effects of off-target expression and improve the efficiency and safety of LVs for gene therapy.

Targeted gene delivery to CD117-expressing cells in vivo with lentiviral vectors co-displaying stem cell factor and a fusogenic molecule.

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody-antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand-receptor recognition mechanism for targeted modification of c-KIT receptor-expressing cells. For targeting, we incorporate membrane-bound human stem cell factor (hSCF), and for fusion, a Sindbis virus-derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF-receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell-specific surface receptors. Development of a high titer lentivector to receptor-specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non-target cells.