Gene therapy for neurotransmitter-related disorders.

Inborn errors of neurotransmitter (NT) metabolism are a group of rare, heterogenous diseases with predominant neurological features, such as movement disorders, autonomic dysfunction, and developmental delay. Clinical overlap with other disorders has led to delayed diagnosis and treatment, and some conditions are refractory to oral pharmacotherapies. Gene therapies have been developed and translated to clinics for paediatric inborn errors of metabolism, with 38 interventional clinical trials ongoing to date. Furthermore, efforts in restoring dopamine synthesis and neurotransmission through viral gene therapy have been developed for Parkinson's disease. Along with the recent European Medicines Agency (EMA) and Medicines and Healthcare Products Regulatory Agency (MHRA) approval of an AAV2 gene supplementation therapy for AADC deficiency, promising efficacy and safety profiles can be achieved in this group of diseases. In this review, we present preclinical and clinical advances to address NT-related diseases, and summarise potential challenges that require careful considerations for NT gene therapy studies.

mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria.

The urea cycle enzyme argininosuccinate lyase (ASL) enables the clearance of neurotoxic ammonia and the biosynthesis of arginine. Patients with ASL deficiency present with argininosuccinic aciduria, an inherited metabolic disease with hyperammonemia and a systemic phenotype coinciding with neurocognitive impairment and chronic liver disease. Here, we describe the dysregulation of glutathione biosynthesis and upstream cysteine utilization in ASL-deficient patients and mice using targeted metabolomics and in vivo positron emission tomography (PET) imaging using (S)-4-(3-18F-fluoropropyl)-l-glutamate ([18F]FSPG). Up-regulation of cysteine metabolism contrasted with glutathione depletion and down-regulated antioxidant pathways. To assess hepatic glutathione dysregulation and liver disease, we present [18F]FSPG PET as a noninvasive diagnostic tool to monitor therapeutic response in argininosuccinic aciduria. Human hASL mRNA encapsulated in lipid nanoparticles improved glutathione metabolism and chronic liver disease. In addition, hASL mRNA therapy corrected and rescued the neonatal and adult Asl-deficient mouse phenotypes, respectively, enhancing ureagenesis. These findings provide mechanistic insights in liver glutathione metabolism and support clinical translation of mRNA therapy for argininosuccinic aciduria.

Fetal gene therapy.

Fetal gene therapy was first proposed toward the end of the 1990s when the field of gene therapy was, to quote the Gartner hype cycle, at its "peak of inflated expectations." Gene therapy was still an immature field but over the ensuing decade, it matured and is now a clinical and market reality. The trajectory of treatment for several genetic diseases is toward earlier intervention. The ability, capacity, and the will to diagnose genetic disease early-in utero-improves day by day. A confluence of clinical trials now signposts a trajectory toward fetal gene therapy. In this review, we recount the history of fetal gene therapy in the context of the broader field, discuss advances in fetal surgery and diagnosis, and explore the full ambit of preclinical gene therapy for inherited metabolic disease.

Systemic gene therapy rescues retinal dysfunction and hearing loss in a model of Norrie disease.

Deafness affects 5% of the world's population, yet there is a lack of treatments to prevent hearing loss due to genetic causes. Norrie disease is a recessive X-linked disorder, caused by NDP gene mutation. It manifests as blindness at birth and progressive sensorineural hearing loss, leading to debilitating dual sensory deprivation. To develop a gene therapy, we used a Norrie disease mouse model (Ndptm1Wbrg ), which recapitulates abnormal retinal vascularisation and progressive hearing loss. We delivered human NDP cDNA by intravenous injection of adeno-associated viral vector (AAV)9 at neonatal, juvenile and young adult pathological stages and investigated its therapeutic effects on the retina and cochlea. Neonatal treatment prevented the death of the sensory cochlear hair cells and rescued cochlear disease biomarkers as demonstrated by RNAseq and physiological measurements of auditory function. Retinal vascularisation and electroretinograms were restored to normal by neonatal treatment. Delivery of NDP gene therapy after the onset of the degenerative inner ear disease also ameliorated the cochlear pathology, supporting the feasibility of a clinical treatment for progressive hearing loss in people with Norrie disease.

Dopamine Transporter Deficiency Syndrome (DTDS): Expanding the Clinical Phenotype and Precision Medicine Approaches.

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy.

Gene Therapy for Dopamine Dyshomeostasis: From Parkinson's to Primary Neurotransmitter Diseases.

Neurological disorders encompass a broad range of neurodegenerative and neurodevelopmental diseases that are complex and almost universally without disease modifying treatments. There is, therefore, significant unmet clinical need to develop novel therapeutic strategies for these patients. Viral gene therapies are a promising approach, where gene delivery is achieved through viral vectors such as adeno-associated virus and lentivirus. The clinical efficacy of such gene therapies has already been observed in two neurological disorders of pediatric onset; for spinal muscular atrophy and aromatic L-amino acid decarboxylase (AADC) deficiency, gene therapy has significantly modified the natural history of disease in these life-limiting neurological disorders. Here, we review recent advances in gene therapy, focused on the targeted delivery of dopaminergic genes for Parkinson's disease and the primary neurotransmitter disorders, AADC deficiency and dopamine transporter deficiency syndrome (DTDS). Although recent European Medicines Agency and Medicines and Healthcare products Regulatory Agency approval of Upstaza (eladocagene exuparvovec) signifies an important landmark, numerous challenges remain. Future research will need to focus on defining the optimal therapeutic window for clinical intervention, better understanding of the duration of therapeutic efficacy, and improved brain targeting. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Microfluidic production of nanogels as alternative triple transfection reagents for the manufacture of adeno-associated virus vectors.

Adeno-associated viral vectors (AAVs) have proved a mainstay in gene therapy, owing to their remarkable transduction efficiency and safety profile. Their production, however, remains challenging in terms of yield, the cost-effectiveness of manufacturing procedures and large-scale production. In this work, we present nanogels produced by microfluidics as a novel alternative to standard transfection reagents such as polyethylenimine-MAX (PEI-MAX) for the production of AAV vectors with comparable yields. Nanogels were formed at pDNA weight ratios of 1 : 1 : 2 and 1 : 1 : 3, of pAAV cis-plasmid, pDG9 capsid trans-plasmid and pHGTI helper plasmid respectively, where vector yields at a small scale showed no significant difference to those of PEI-MAX. Weight ratios of 1 : 1 : 2 showed overall higher titers than 1 : 1 : 3, where nanogels with nitrogen/phosphate ratios of 5 and 10 produced yields of ≈8.8 × 108 vg mL-1 and ≈8.1 × 108 vg mL-1 respectively compared to ≈1.1 × 109 vg mL-1 for PEI-MAX. In larger scale production, optimised nanogels produced AAV at a titer of ≈7.4 × 1011 vg mL-1, showing no statistical difference from that of PEI-MAX at ≈1.2 × 1012 vg mL-1, indicating that equivalent titers can be achieved with easy-to-implement microfluidic technology at comparably lower costs than traditional reagents.

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival.

Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.

Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology.

Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at -20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.

Safety and efficacy of an engineered hepatotropic AAV gene therapy for ornithine transcarbamylase deficiency in cynomolgus monkeys.

X-linked inherited ornithine transcarbamylase deficiency (OTCD) is the most common disorder affecting the liver-based urea cycle, a pathway enabling detoxification of nitrogen waste and endogenous arginine biosynthesis. Patients develop acute hyperammonemia leading to neurological sequelae or death despite the best-accepted therapy based on ammonia scavengers and protein-restricted diet. Liver transplantation is curative but associated with procedure-related complications and lifelong immunosuppression. Adeno-associated viral (AAV) vectors have demonstrated safety and clinical benefits in a rapidly growing number of clinical trials for inherited metabolic liver diseases. Engineered AAV capsids have shown promising enhanced liver tropism. Here, we conducted a good-laboratory practice-compliant investigational new drug-enabling study to assess the safety of intravenous liver-tropic AAVLK03 gene transfer of a human codon-optimized OTC gene. Juvenile cynomolgus monkeys received vehicle and a low and high dose of vector (2 × 1012 and 2 × 1013 vector genome (vg)/kg, respectively) and were monitored for 26 weeks for in-life safety with sequential liver biopsies at 1 and 13 weeks post-vector administration. Upon completion of monitoring, animals were euthanized to study vector biodistribution, immune responses, and histopathology. The product was well tolerated with no adverse clinical events, predominant hepatic biodistribution, and sustained supra-physiological OTC overexpression. This study supports the clinical deployment of intravenous AAVLK03 for severe OTCD.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications.

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Gene Therapy for Lysosomal Storage Disorders: Ongoing Studies and Clinical Development.

Rare monogenic disorders such as lysosomal diseases have been at the forefront in the development of novel treatments where therapeutic options are either limited or unavailable. The increasing number of successful pre-clinical and clinical studies in the last decade demonstrates that gene therapy represents a feasible option to address the unmet medical need of these patients. This article provides a comprehensive overview of the current state of the field, reviewing the most used viral gene delivery vectors in the context of lysosomal storage disorders, a selection of relevant pre-clinical studies and ongoing clinical trials within recent years.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation.

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.

Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders.

Urea cycle disorders (UCD) are inherited defects in clearance of waste nitrogen with high morbidity and mortality. Novel and more effective therapies for UCD are needed. Studies in mice with constitutive activation of autophagy unravelled Beclin-1 as druggable candidate for therapy of hyperammonemia. Next, we investigated efficacy of cell-penetrating autophagy-inducing Tat-Beclin-1 (TB-1) peptide for therapy of the two most common UCD, namely ornithine transcarbamylase (OTC) and argininosuccinate lyase (ASL) deficiencies. TB-1 reduced urinary orotic acid and improved survival under protein-rich diet in spf-ash mice, a model of OTC deficiency (proximal UCD). In AslNeo/Neo mice, a model of ASL deficiency (distal UCD), TB-1 increased ureagenesis, reduced argininosuccinate, and improved survival. Moreover, it alleviated hepatocellular injury and decreased both cytoplasmic and nuclear glycogen accumulation in AslNeo/Neo mice. In conclusion, Beclin-1-dependent activation of autophagy improved biochemical and clinical phenotypes of proximal and distal defects of the urea cycle.

Cervical Gene Delivery of the Antimicrobial Peptide, Human ß-Defensin (HBD)-3, in a Mouse Model of Ascending Infection-Related Preterm Birth.

Approximately 40% of preterm births are preceded by microbial invasion of the intrauterine space; ascent from the vagina being the most common pathway. Within the cervical canal, antimicrobial peptides and proteins (AMPs) are important components of the cervical barrier which help to prevent ascending vaginal infection. We investigated whether expression of the AMP, human ß-defensin-3 (HBD3), in the cervical mucosa of pregnant mice could prevent bacterial ascent from the vagina into the uterine cavity. An adeno-associated virus vector containing both the HBD3 gene and GFP transgene (AAV8 HBD3.GFP) or control AAV8 GFP, was administered intravaginally into E13.5 pregnant mice. Ascending infection was induced at E16.5 using bioluminescent Escherichia coli (E. coli K1 A192PP-lux2). Bioluminescence imaging showed bacterial ascent into the uterine cavity, inflammatory events that led to premature delivery and a reduction in pups born alive, compared with uninfected controls. Interestingly, a significant reduction in uterine bioluminescence in the AAV8 HBD3.GFP-treated mice was observed 24 h post-E. coli infection, compared to AAV8 GFP treated mice, signifying reduced bacterial ascent in AAV8 HBD3.GFP-treated mice. Furthermore, there was a significant increase in the number of living pups in AAV HBD3.GFP-treated mice. We propose that HBD3 may be a potential candidate for augmenting cervical innate immunity to prevent ascending infection-related preterm birth and its associated neonatal consequences.

Correction: Impaired cellular bioenergetics caused by GBA1 depletion sensitizes neurons to calcium overload.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes.

Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.