Gene and cell therapy of human genetic diseases: Recent advances and future directions.

Disruptions in normal development and the emergence of health conditions often result from the malfunction of vital genes in the human body. Decades of scientific research have focused on techniques to modify or substitute defective genes with healthy alternatives, marking a new era in disease treatment, prevention and cure. Recent strides in science and technology have reshaped our understanding of disorders, medication development and treatment recommendations, with human gene and cell therapy at the forefront of this transformative shift. Its primary objective is the modification of genes or adjustment of cell behaviour for therapeutic purposes. In this review, we focus on the latest advances in gene and cell therapy for treating human genetic diseases, with a particular emphasis on FDA and EMA-approved therapies and the evolving landscape of genome editing. We examine the current state of innovative gene editing technologies, particularly the CRISPR-Cas systems. As we explore the progress, ethical considerations and prospects of these innovations, we gain insight into their potential to revolutionize the treatment of genetic diseases, along with a discussion of the challenges associated with their regulatory pathways. This review traces the origins and evolution of these therapies, from conceptual ideas to practical clinical applications, marking a significant milestone in the field of medical science.

Comparing Viral Vectors and Fate Mapping Approaches for Astrocyte-to-Neuron Reprogramming in the Injured Mouse Cerebral Cortex.

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Advances in delivery systems for CRISPR/Cas-mediated cancer treatment: a focus on viral vectors and extracellular vesicles.

The delivery of CRISPR/Cas systems holds immense potential for revolutionizing cancer treatment, with recent advancements focusing on extracellular vesicles (EVs) and viral vectors. EVs, particularly exosomes, offer promising opportunities for targeted therapy due to their natural cargo transport capabilities. Engineered EVs have shown efficacy in delivering CRISPR/Cas components to tumor cells, resulting in inhibited cancer cell proliferation and enhanced chemotherapy sensitivity. However, challenges such as off-target effects and immune responses remain significant hurdles. Viral vectors, including adeno-associated viruses (AAVs) and adenoviral vectors (AdVs), represent robust delivery platforms for CRISPR/Cas systems. AAVs, known for their safety profile, have already been employed in clinical trials for gene therapy, demonstrating their potential in cancer treatment. AdVs, capable of infecting both dividing and non-dividing cells, offer versatility in CRISPR/Cas delivery for disease modeling and drug discovery. Despite their efficacy, viral vectors present several challenges, including immune responses and off-target effects. Future directions entail refining delivery systems to enhance specificity and minimize adverse effects, heralding personalized and effective CRISPR/Cas-mediated cancer therapies. This article underscores the importance of optimized delivery mechanisms in realizing the full therapeutic potential of CRISPR/Cas technology in oncology. As the field progresses, addressing these challenges will be pivotal for translating CRISPR/Cas-mediated cancer treatments from bench to bedside.

How Advanced are Nanocarriers for Effective Subretinal Injection?

Subretinal injection (SR injection) is a commonly used method of ocular drug delivery and has been mainly applied for the treatment of neovascular age-associated macular degeneration (nAMD) and sub-macular hemorrhage (SMH) caused by nAMD, as well as various types of hereditary retinopathies (IRD) such as Stargardt's disease (STGD), retinitis pigmentosa (RP), and a series of fundus diseases such as Leber's congenital dark haze (LCA), choroidal defects, etc. The commonly used carriers of SR injection are mainly divided into viral and non-viral vectors. Leber's congenital amaurosis (LCA), choroidal agenesis, and a series of other fundus diseases are also commonly treated using SR injection. The commonly used vectors for SR injection are divided into two categories: viral vectors and non-viral vectors. Viral vectors are a traditional class of SR injection drug carriers that have been extensively studied in clinical treatment, but they still have many limitations that cannot be ignored, such as poor reproduction efficiency, small loading genes, and triggering of immune reactions. With the rapid development of nanotechnology in the treatment of ocular diseases, nanovectors have become a research hotspot in the field of non-viral vectors. Nanocarriers have numerous attractive properties such as low immunogenicity, robust loading capacity, stable structure, and easy modification. These valuable features imply greater safety, improved therapeutic efficacy, longer duration, and more flexible indications. In recent years, there has been a growing interest in nanocarriers, which has led to significant advancements in the treatment of ocular diseases. Nanocarriers have not only successfully addressed clinical problems that viral vectors have failed to overcome but have also introduced new therapeutic possibilities for certain classical disease types. Nanocarriers offer undeniable advantages over viral vectors. This review discusses the advantages of subretinal (SR) injection, the current status of research, and the research hotspots of gene therapy with viral vectors. It focuses on the latest progress of nanocarriers in SR injection and enumerates the limitations and future perspectives of nanocarriers in the treatment of fundus lesions. Furthermore, this review also covers the research progress of nanocarriers in the field of subretinal injection and highlights the value of nanocarrier-mediated SR injection in the treatment of fundus disorders. Overall, it provides a theoretical basis for the application of nanocarriers in SR injection.

Lipid-based Non-viral Vector: Promising Approach for Gene Delivery.

OBJECTIVES: The present review aims to discuss various strategies to overcome intracellular and extracellular barriers involved in gene delivery as well as the advantages, challenges, and mechanisms of gene delivery using non-viral vectors. Additionally, patents, clinical studies, and various formulation approaches related to lipid-based carrier systems are discussed. METHODS: Data were searched and collected from Google Scholar, ScienceDirect, Pubmed, and Springer. RESULTS: In this review, we have investigated the advantages of non-viral vectors over viral vectors. The advantage of using non-viral vectors are that they seek more attention in different fields. They play an important role in delivering the genetic materials. However, few nonviral vector-based carrier systems have been found in clinical settings. Challenges are developing more stable, site-specific gene delivery and conducting thorough safety assessments to minimize the undesired effects. CONCLUSION: In comparison to viral vectors, nonviral vector-based lipid nanocarriers have more advantages for gene delivery. Gene therapy research shows promise in addressing health concerns. Lipid-based nanocarriers can overcome intracellular and extracellular barriers, allowing efficient delivery of genetic materials. Non-viral vectors are more attractive due to their biocompatibility, ease of synthesis, and cost-effectiveness. They can deliver various nucleic acids and have improved gene delivery efficacy by avoiding degradation steps. Despite limited clinical use, many patents have been filed for mRNA vaccine delivery using non-viral vectors.

Immunogenicity risk assessment of empty capsids present in adeno-associated viral vectors using predictive innate immune responses.

Immunogenicity of gene therapy and the impacts on safety and efficacy are of increasing interest in the pharmaceutical industry. Unique structural aspects of gene therapy delivery vectors, such as adeno-associated viral (AAV) vectors, are expected to activate the innate immune system. The risk of innate immune activation is critical to understand due to the potential impacts on safety and on subsequent adaptive immune responses. In this study, we investigated the responses of key innate immune players-dendritic cells, natural killer (NK) cells, and the complement system-to AAV8 capsids. Immunogenicity risk was also predicted in the presence empty AAV capsids for AAV gene therapy. Compared to genome-containing "full" AAV8 capsids, empty AAV8 capsids more strongly induced proinflammatory cytokine production and migration by human and mouse dendritic cells, but the "full" capsid increased expression of co-stimulatory markers. Furthermore, in an NK cell degranulation assay, we found mixtures of empty and full AAV8 capsids to activate expression of TNF-α, IFN-γ, and CD107a more strongly in multiple NK cell populations compared to either capsid type alone. Serum complement C3a was also induced more strongly in the presence of mixed empty and full AAV8 capsid formulations. Risk for innate immune activation suggests the importance to determine acceptable limits of empty capsids. Immunogenicity risk assessment of novel biological modalities will benefit from the aforementioned in vitro innate immune activation assays providing valuable mechanistic information.

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

Smoldering oncolysis by foamy virus carrying CD19 as a CAR target escapes CAR T detection by genomic modification.

Chimeric antigen receptor (CAR) T cells have had limited success against solid tumors. Here, we used an oncolytic foamy virus (oFV) to display a model CAR target antigen (CD19) on tumors in combination with anti-CD19 CAR T cells. We generated oFV-Δbel2 and oFV-bel2 vectors to test the efficiency and stability of viral/CD19 spread. While both viruses conferred equal CAR T killing in vitro, the oFV-Δbel2 virus acquired G-to-A mutations, whereas oFV-bel2 virus had genome deletions. In subcutaneous tumor models in vivo, CAR T cells led to a significant decrease in oFV-specific bioluminescence, confirming clearance of oFV-infected tumor cells. However, the most effective therapy was with high-dose oFV in the absence of CAR T cells, indicating that CAR T clearance of oFV was detrimental. Moreover, in tumors that escaped CAR T cell treatment, resurgent virus contained deletions within the oFV-CD19 transgene, allowing the virus to escape CAR T elimination. Therefore, oFV represents a slow smoldering type of oncolytic virus, whose chronic spread through tumors generates anti-tumor therapy, which is abolished by CAR T therapy. These results suggest that further development of this oncolytic platform, with additional immunotherapeutic arming, may allow for an effective combination of chronic oncolysis.

In vivo liver targeted genome editing as therapeutic approach: progresses and challenges.

The liver is an essential organ of the body that performs several vital functions, including the metabolism of biomolecules, foreign substances, and toxins, and the production of plasma proteins, such as coagulation factors. There are hundreds of genetic disorders affecting liver functions and, for many of them, the only curative option is orthotopic liver transplantation, which nevertheless entails many risks and long-term complications. Some peculiar features of the liver, such as its large blood flow supply and the tolerogenic immune environment, make it an attractive target for in vivo gene therapy approaches. In recent years, several genome-editing tools mainly based on the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system have been successfully exploited in the context of liver-directed preclinical or clinical therapeutic applications. These include gene knock-out, knock-in, activation, interference, or base and prime editing approaches. Despite many achievements, important challenges still need to be addressed to broaden clinical applications, such as the optimization of the delivery methods, the improvement of the editing efficiency, and the risk of on-target or off-target unwanted effects and chromosomal rearrangements. In this review, we highlight the latest progress in the development of in vivo liver-targeted genome editing approaches for the treatment of genetic disorders. We describe the technological advancements that are currently under investigation, the challenges to overcome for clinical applicability, and the future perspectives of this technology.

The Current Landscape of Secondary Malignancies after CAR T-Cell Therapies: How Could Malignancies Be Prevented?

Chimeric antigen receptor (CAR) T-cell therapies have revolutionised the field of haematological malignancies by achieving impressive remission rates in patients with highly refractory haematological malignancies, improving overall survival. To date, six commercial anti-CD19 and anti-BCMA CAR T-cell products have been approved by the Food and Drug Administration (FDA) for the treatment of relapsed/refractory B-cell haematological malignancies and multiple myeloma. The indications for CAR T-cell therapies are gradually expanding, with these therapies being investigated in a variety of diseases, including non-malignant ones. Despite the great success, there are several challenges surrounding CAR T-cell therapies, such as non-durable responses and high-grade toxicities. In addition, a new safety concern was added by the FDA on 28 November 2023 following reports of T-cell malignancies in patients previously treated with either anti-CD19 or anti-BCMA autologous CAR T-cell therapies both in clinical trials and in the real-world setting. Since then, several reports have been published presenting the incidence and analysing the risks of other secondary malignancies after CAR T-cell therapies. In this opinion article, the current landscape of secondary malignancies after CAR T-cell therapies is presented, along with a proposed strategy for future research aiming at potentially diminishing or abrogating the risk of developing secondary malignancies after CAR T-cell therapies.

Neurosurgical gene therapy for central nervous system diseases.

Viral vector mediated gene therapies for neurodegenerative and neurodevelopmental conditions that require neurosurgical administration continue to expand. We systematically reviewed the National Institutes of Health (NIH) ClinicalTrials.gov database to identify all clinical trials studying in-vivo viral vector mediated gene therapies targeted to the CNS for neurodegenerative and neurodevelopmental diseases. We isolated studies which delivered therapies using neurosurgical approaches: intracisternal, intraventricular, and/or intraparenchymal. Clinical trials primarily registered in international countries were included if they were referenced by an NIH registered clinical trial. We performed a scoping review to identify the preclinical studies that supported each human clinical trial. Key preclinical and clinical data were aggregated to characterize vector capsid design, delivery methods, gene expression profile, and clinical benefit. A total of 64 clinical trials were identified in active, completed, terminated, and long-term follow-up stages. A range of CNS conditions across pediatric and adult populations are being studied with CNS targeted viral vector gene therapy, including Alzheimer's disease, Parkinson's disease, AADC deficiency, sphingolipidoses, mucopolysaccharidoses, neuronal ceroid lipofuscinoses, spinal muscular atrophy, adrenoleukodystrophy, Canavan disease, frontotemporal dementia, Huntington's disease, Rett syndrome, Dravet syndrome, mesial temporal lobe epilepsy, and glutaric acidemia. Adeno-associated viral vectors (AAVs) were utilized by the majority of tested therapies, with vector serotypes, regulatory elements, delivery methods, and vector monitoring varying based on the disease being studied. Intraparenchymal delivery has evolved significantly, with MRI-guided convection-enhanced delivery established as a gold standard method for pioneering novel gene targets.

Formulation of three tailed bacteriophages by spray-drying and atomic layer deposition for thermal stability and controlled release.

Deep infection is the second most common complication of arthroplasty following loosening of the implant. Antibiotic-loaded bone cements (ALBCs) and high concentrations of systemic broad-spectrum antibiotics are commonly used to prevent infections following injury and surgery. However, clinical data fails to show that ALBCs are effective against deep infection, and negative side effects can result following prolonged administration of antibiotics. Additionally, the rise of multidrug resistant (MDR) bacteria provides an urgent need for alternatives to broad-spectrum antibiotics. Phage therapy, or the use of bacteriophages (viruses that infect bacteria) to target pathogenic bacteria, might offer a safe alternative to combat MDR bacteria. Application of phage therapy in the setting of deep infections requires formulation strategies that would stabilize bacteriophage against chemical and thermal stress during bone-cement polymerization, that maintain bacteriophage activity for weeks or months at physiological temperatures, and that allow for sustained release of phage to combat slow-growing, persistent bacteria. Here, we demonstrate the formulation of three phages that target diverse bacterial pathogens, which includes spray-drying of the particles for enhanced thermal stability at 37 °C and above. Additionally, we use atomic layer deposition (ALD) to coat spray-dried powders with alumina to allow for delayed release of phage from the dry formulations, and potentially protect phage against chemical damage during bone cement polymerization. Together, these findings present a strategy to formulate phages that possess thermal stability and sustained release properties for use in deep infections.

A Simple and Versatile Method for Ex Vivo Monitoring of Goat Vaginal Mucosa Transduction by Viral Vector Vaccines.

Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector's ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2's ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases.

T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

Viral vector-based therapeutic HPV vaccines.

Replication-defective viral vector vaccines have several advantages over conventional subunit vaccines, including potent antibody responses, cellular responses critical for eliminating pathogen-infected cells, and the induction of highly immunogenic and durable immune responses without adjuvants. The Human papillomavirus (HPV), a microorganism with over 200 genotypes, plays a crucial role in inducing human tumors, with the majority of HPV-related malignancies expressing HPV proteins. Tumors associated with HPV infection, most of which result from HPV16 infection, include those affecting the cervix, anus, vagina, penis, vulva, and oropharynx. In recent years, the development of therapeutic HPV vaccines utilizing viral vectors for the treatment of premalignant lesions or tumors caused by HPV infection has experienced rapid growth, with numerous research pipelines currently underway. Simultaneously, screening for optimal antigens requires more basic research and more optimized methods. In terms of preclinical research, we present the various models used to assess vaccine efficacy, highlighting their respective advantages and disadvantages. Further, we present current research status of therapeutic vaccines using HPV viral vectors, especially the indications, initial efficacy, combination drugs, etc. In general, this paper summarizes current viral vector therapeutic HPV vaccines in terms of HPV infection, antigen selection, vectors, efficacy evaluation, and progress in clinical trials.

Endothelial-to-mesenchymal transition enhances permissiveness to AAV vectors in cardiac endothelial cells.

A major obstacle in inducing therapeutic angiogenesis in the heart is inefficient gene transfer to endothelial cells (ECs). Here, we identify compounds able to enhance the permissiveness of cardiac ECs to adeno-associated virus (AAV) vectors, which stand as ideal tools for in vivo gene delivery. We screened a library of >1,500 US Food and Drug Administration (FDA)-approved drugs, in combination with AAV vectors, in cardiac ECs. Among the top drugs increasing AAV-mediated transduction, we found vatalanib, an inhibitor of multiple tyrosine kinase receptors. The increased AAV transduction efficiency by vatalanib was paralleled by induction of the endothelial-to-mesenchymal transition, as documented by decreased endothelial and increased mesenchymal marker expression. Induction of the endothelial-to-mesenchymal transition by other strategies similarly increased EC permissiveness to AAV vectors. In vivo injection of AAV vectors in the heart after myocardial infarction resulted in the selective transduction of cells undergoing the endothelial-to-mesenchymal transition, which is known to happen transiently after cardiac ischemia. Collectively, these results point to the endothelial-to-mesenchymal transition as a mechanism for improving AAV transduction in cardiac ECs, with implications for both basic research and the induction of therapeutic angiogenesis in the heart.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems.

Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.

Systemic delivery of AAV-GCDH ameliorates HLD-induced phenotype in a glutaric aciduria type I mouse model.

Glutaric aciduria type 1 (GA1) is a rare inherited metabolic disorder caused by a deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), with accumulation of neurotoxic metabolites, resulting in a complex movement disorder, irreversible brain damage, and premature death in untreated individuals. While early diagnosis and a lysine restricted diet can extend survival, they do not prevent neurological damage in approximately one-third of treated patients, and more effective therapies are required. Here we report the efficacy of adeno-associated virus 9 (AAV9)-mediated systemic delivery of human GCDH at preventing a high lysine diet (HLD)-induced phenotype in Gcdh -/- mice. Neonatal treatment with AAV-GCDH restores GCDH expression and enzyme activity in liver and striatum. This treatment protects the mice from HLD-aggressive phenotype with all mice surviving this exposure; in stark contrast, a lack of treatment on an HLD triggers very high accumulation of glutaric acid, 3-hydroxyglutaric acid, and glutarylcarnitine in tissues, with about 60% death due to brain accumulation of toxic lysine metabolites. AAV-GCDH significantly ameliorates the striatal neuropathology, minimizing neuronal dysfunction, gliosis, and alterations in myelination. Magnetic resonance imaging findings show protection against striatal injury. Altogether, these results provide preclinical evidence to support AAV-GCDH gene therapy for GA1.

Recent Advances in Gene Therapy for Hemophilia: Projecting the Perspectives.

One of the well-known X-linked genetic disorders is hemophilia, which could be hemophilia A as a result of a mutation in the F8 (factor VIII) gene or hemophilia B as a result of a mutation in the F9 (factor IX) gene, leading to insufficient levels of the proteins essential for blood coagulation cascade. In patients with severe hemophilia, factor VIII or factor IX activities in the blood plasma are considerably low, estimated to be less than 1%. This is responsible for spontaneous or post-traumatic bleeding episodes, or both, leading to disease complications and death. Current treatment of hemophilia relies on the prevention of bleeding, which consists of expensive lifelong replacement infusion therapy of blood plasma clotting factors, their recombinant versions, or therapy with recombinant monoclonal antibodies. Recently emerged gene therapy approaches may be a potential game changer that could reshape the therapeutic outcomes of hemophilia A or B using a one-off vector in vivo delivery and aim to achieve long-term endogenous expression of factor VIII or IX. This review examines both traditional approaches to the treatment of hemophilia and modern methods, primarily focusing on gene therapy, to update knowledge in this area. Recent technological advances and gene therapeutics in the pipeline are critically reviewed and summarized. We consider gene therapy to be the most promising method as it may overcome the problems associated with more traditional treatments, such as the need for constant and expensive infusions and the presence of an immune response to the antibody drugs used to treat hemophilia.