Peripheral expression of brain-penetrant progranulin rescues pathologies in mouse models of frontotemporal lobar degeneration.

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TAR DNA-binding protein 43 (TDP-43) pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN in the CNS, including gene therapy. However, a limitation of current gene therapy approaches aimed to alleviate FTLD-associated pathologies may be their inefficient brain exposure and biodistribution. We therefore developed an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant [AAV(L):bPGRN] in two mouse models of FTLD-GRN, namely, Grn knockout and GrnxTmem106b double knockout mice. This therapeutic strategy avoids potential safety and biodistribution issues of CNS-administered AAVs and maintains sustained concentrations of PGRN in the brain after a single dose. AAV(L):bPGRN treatment reduced several FTLD-GRN-associated pathologies including severe motor function deficits, aberrant TDP-43 phosphorylation, dysfunctional protein degradation, lipid metabolism, gliosis, and neurodegeneration in the brain. The potential translatability of our findings was tested in an in vitro model using cocultured human induced pluripotent stem cell (hiPSC)-derived microglia lacking PGRN and TMEM106B and wild-type hiPSC-derived neurons. As in mice, aberrant TDP-43, lysosomal dysfunction, and neuronal loss were ameliorated after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN). Together, our studies suggest that peripherally administered brain-penetrant PGRN replacement strategies ameliorate FTLD-GRN relevant phenotypes including TDP-43 pathology, neurodegeneration, and behavioral deficits. Our data provide preclinical proof of concept for the use of this AAV platform for treatment of FTLD-GRN and potentially other CNS disorders.

Recent progress of iron-based nanomaterials in gene delivery and tumor gene therapy.

Gene therapy aims to modify or manipulate gene expression and change the biological characteristics of living cells to achieve the purpose of treating diseases. The safe, efficient, and stable expression of exogenous genes in cells is crucial for the success of gene therapy, which is closely related to the vectors used in gene therapy. Currently, gene therapy vectors are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors are widely used due to the advantages of persistent and stable expression, high transfection efficiency, but they also have certain issues such as infectivity, high immunological rejection, randomness of insertion mutation, carcinogenicity, and limited vector capacity. Non-viral vectors have the advantages of non-infectivity, controllable chemical structure, and unlimited vector capacity, but the transfection efficiency is low. With the rapid development of nanotechnology, the unique physicochemical properties of nanomaterials have attracted increasing attention in the field of drug and gene delivery. Among many nanomaterials, iron-based nanomaterials have attracted much attention due to their superior physicochemical properties, such as Fenton reaction, magnetic resonance imaging, magnetothermal therapy, photothermal therapy, gene delivery, magnetically-assisted drug delivery, cell and tissue targeting, and so on. In this paper, the research progress of iron-based nanomaterials in gene delivery and tumor gene therapy is reviewed, and the future application direction of iron-based nanomaterials is further prospected.

Revolutionizing Lung Cancer Treatment: Innovative CRISPR-Cas9 Delivery Strategies.

Lung carcinoma, including both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), remains a significant global health challenge due to its high morbidity and mortality rates. The objsective of this review is to meticulously examine the current advancements and strategies in the delivery of CRISPR-Cas9 gene-editing technology for the treatment of lung carcinoma. This technology heralds a new era in molecular biology, offering unprecedented precision in genomic modifications. However, its therapeutic potential is contingent upon the development of effective delivery mechanisms that ensure the efficient and specific transport of gene-editing tools to tumor cells. We explore a variety of delivery approaches, such as viral vectors, lipid-based nanoparticles, and physical methods, highlighting their respective advantages, limitations, and recent breakthroughs. This review also delves into the translational and clinical significance of these strategies, discussing preclinical and clinical studies that investigate the feasibility, efficacy, and safety of CRISPR-Cas9 delivery for lung carcinoma. By scrutinizing the landscape of ongoing clinical trials and offering translational perspectives, we aim to elucidate the current state and future directions of this rapidly evolving field. The review is structured to first introduce the problem and significance of lung carcinoma, followed by an overview of CRISPR-Cas9 technology, a detailed examination of delivery strategies, and an analysis of clinical applications and regulatory considerations. Our discussion concludes with future perspectives and challenges, such as optimizing delivery strategies, enhancing specificity, mitigating immunogenicity concerns, and addressing regulatory issues. This comprehensive overview seeks to provide insights into the potential of CRISPR-Cas9 as a revolutionary approach for targeted therapies and personalized medicine in lung carcinoma, emphasizing the importance of delivery strategy development in realizing the full potential of this groundbreaking technology.

Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype.

The microtubule-associated protein Tau is a key player in various neurodegenerative conditions, including Alzheimer's disease (AD) and Tauopathies, where its hyperphosphorylation disrupts neuronal microtubular lattice stability. Glaucoma, a neurodegenerative disorder affecting the retina, leads to irreversible vision loss by damaging retinal ganglion cells and the optic nerve, often associated with increased intraocular pressure. Prior studies have indicated Tau expression and phosphorylation alterations in the retina in both AD and glaucoma, yet the causative or downstream nature of Tau protein changes in these pathologies remains unclear. This study investigates the impact of Tau protein modulation on retinal neurons under normal and experimental glaucoma conditions. Employing AAV9-mediated gene therapy for Tau overexpression and knockdown, both manipulations were found to adversely affect retinal structural and functional measures as well as neuroprotective Akt/Erk survival signalling in healthy conditions. In the experimental glaucoma model, Tau overexpression intensified inner retinal degeneration, while Tau silencing provided significant protection against these degenerative changes. These findings underscore the critical role of endogenous Tau protein levels in preserving retinal integrity and emphasize the therapeutic potential of targeting Tau in glaucoma pathology.

Gene therapy for people with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the most common type of liver cancer, accounting for 70% to 85% of individuals with primary liver cancer. Gene therapy, which uses genes to treat or prevent diseases, holds potential for treatment, especially for tumours. Trials on the effects of gene therapy in people with hepatocellular carcinoma have been published or are ongoing. OBJECTIVES: To evaluate the benefits and harms of gene therapy in people with hepatocellular carcinoma, irrespective of sex, administered dose, and type of formulation. SEARCH METHODS: We identified randomised clinical trials through electronic searches in The Cochrane Hepato-Biliary Group Controlled Trials Register, CENTRAL, MEDLINE, Embase, LILACS, Science Citation Index Expanded, and Conference Proceedings Citation Index-Science. We searched five online clinical trial registries to identify unpublished or ongoing trials. We checked reference lists of the retrieved studies for further trials. The date of last search was 20 January 2023. SELECTION CRITERIA: We aimed to include randomised clinical trials assessing any type of gene therapy in people diagnosed with hepatocellular carcinoma, irrespective of year, language of publication, format, or outcomes reported. DATA COLLECTION AND ANALYSIS: We followed Cochrane methodology and used Review Manager to prepare the review. The primary outcomes were all-cause mortality/overall survival (whatever data were provided), serious adverse events during treatment, and health-related quality of life. The secondary outcomes were proportion of people with disease progression, adverse events considered non-serious, and proportion of people without improvement in liver function tests. We assessed risk of bias of the included trials using RoB 2 and the certainty of evidence using GRADE. We presented the results of time-to-event outcomes as hazard ratios (HR), dichotomous outcomes as risk ratios (RR), and continuous outcomes as mean difference (MD) with their 95% confidence intervals (CI). Our primary analyses were based on intention-to-treat and outcome data at the longest follow-up. MAIN RESULTS: We included six randomised clinical trials with 364 participants. The participants had unresectable (i.e. advanced inoperable) hepatocellular carcinoma. We found no trials assessing the effects of gene therapy in people with operable hepatocellular carcinoma. Four trials were conducted in China, one in several countries (from North America, Asia, and Europe), and one in Egypt. The number of participants in the six trials ranged from 10 to 129 (median 47), median age was 55.2 years, and the mean proportion of males was 72.7%. The follow-up duration ranged from six months to five years. As the trials compared different types of gene therapy and had different controls, we could not perform meta-analyses. Five of the six trials administered co-interventions equally to the experimental and control groups. All trials assessed one or more outcomes of interest in this review. The certainty of evidence was very low in five of the six comparisons and low in the double-dose gene therapy comparison. Below, we reported the results of the primary outcomes only. Pexastimogene devacirepvec (Pexa-Vec) plus best supportive care versus best supportive care alone There is uncertainty about whether there may be little to no difference between the effect of Pexa-Vec plus best supportive care compared with best supportive care alone on overall survival (HR 1.19, 95% CI 0.78 to 1.82; 1 trial (censored observation at 20-month follow-up), 129 participants; very low-certainty evidence) and on serious adverse events (RR 1.42, 95% CI 0.60 to 3.33; 1 trial at 20 months after treatment, 129 participants; very low-certainty evidence). The trial reported quality of life narratively as "assessment of quality of life and time to symptomatic progression was confounded by the high patient dropout rate." Adenovirus-thymidine kinase with ganciclovir (ADV-TK/GCV) plus liver transplantation versus liver transplantation alone There is uncertainty about whether ADV-TK/GCV plus liver transplantation may benefit all-cause mortality at the two-year follow-up (RR 0.39, 95% CI 0.20 to 0.76; 1 trial, 45 participants; very low-certainty evidence). The trial did not report serious adverse events other than mortality or quality of life. Double-dose ADV-TK/GCV plus liver transplantation versus liver transplantation alone There is uncertainty about whether double-dose ADV-TK/GCV plus liver transplantation versus liver transplantation may benefit all-cause mortality at five-year follow-up (RR 0.40, 95% CI 0.22 to 0.73; 1 trial, 86 participants; low-certainty evidence). The trial did not report serious adverse events other than mortality or quality of life. Recombinant human adenovirus-p53 with hydroxycamptothecin (rAd-p53/HCT) versus hydroxycamptothecin alone There is uncertainty about whether there may be little to no difference between the effect of rAd-p53/HCT versus hydroxycamptothecin alone on the overall survival at 12-month follow-up (RR 3.06, 95% CI 0.16 to 60.47; 1 trial, 48 participants; very low-certainty evidence). The trial did not report serious adverse events or quality of life. rAd-p53/5-Fu (5-fluorouracil) plus transarterial chemoembolisation versus transarterial chemoembolisation alone The trial included 46 participants. We had insufficient data to assess overall survival. The trial did not report serious adverse events or quality of life. E1B-deleted (dl1520) adenovirus versus percutaneous ethanol injection The trial included 10 participants. It did not report data on overall survival, serious adverse events, or health-related quality of life. One trial did not provide any information on sponsorship; one trial received a national research grant, one trial by the Pedersen foundation, and three were industry-funded trials. We found five ongoing randomised clinical trials. AUTHORS' CONCLUSIONS: The evidence is very uncertain about the effects of gene therapy on the studied outcomes because of high risk of bias and imprecision of outcome results. The trials were underpowered and lacked trial data on clinically important outcomes. There was only one trial per comparison, and we could not perform meta-analyses. Therefore, we do not know if gene therapy may reduce, increase, or have little to no effect on all-cause mortality or overall survival, or serious adverse events in adults with unresectable hepatocellular carcinoma. The impact of gene therapy on adverse events needs to be investigated further. Evidence on the effect of gene therapy on health-related quality of life is lacking.

The oncogenic roles of GPR176 in ovarian cancer: a molecular target for aggressiveness and gene therapy.

BACKGROUND: At present, the discovery of new biomarkers is of great significance for the early diagnosis, treatment and prognosis assessment of ovarian cancer. Previous findings indicated that aberrant G-protein-coupled receptor 176 (GPR176) expression might contribute to tumorigenesis and subsequent progression. However, the expression of GPR176 and the molecular mechanisms in ovarian cancer had not been investigated. METHODS: GPR176 expression was compared with clinicopathological features of ovarian cancer using immunohistochemical and bioinformatics analyses. GPR176-related genes and pathways were analysed using bioinformatics analysis. Additionally, the effects of GPR176 on ovarian cancer cell phenotypes were investigated. RESULTS: GPR176 expression positively correlated with elder age, clinicopathological staging, tumour residual status, and unfavourable survival of ovarian cancer, but negatively with purity loss, infiltration of B cells, and CD8+ T cells. Gene Set Enrichment Analysis showed that differential expression of GPR176 was involved in focal adhesion, ECM-receptor interaction, cell adhesion molecules and so on. STRING and Cytoscape were used to determine the top 10 nodes. Kyoto Encyclopaedia of Genes and Genomes analysis indicated that GPR176-related genes were involved in the ECM structural constituent and organisation and so on. GPR176 overexpression promoted the proliferation, anti-apoptosis, anti-pyroptosis, migration and invasion of ovarian cancer cells with overexpression of N-cadherin, Zeb1, Snail, Twist1, and under-expression of gasdermin D, caspase 1, and E-cadherin. CONCLUSION: GPR176 might be involved in the progression of ovarian cancer. It might be used as a biomarker to indicate the aggressive behaviour and poor prognosis of ovarian cancer and a target of genetic therapy.

Ovarian cancer is a gynecological cancer with high mortality. Due to the limited screening tests and treatments available, most ovarian cancer patients are diagnosed at a late stage and the prognosis is poor. The addition of new cancer diagnostic biomarkers and new intervention targets may improve quality of life and survival for patients with ovarian cancer. Previous studies have revealed the aberrant GPR176 expression might contribute to tumorigenesis and subsequent progression in many other tumours. In our study, GPR176 was found to promote the proliferation, anti-apoptosis, anti-pyroptosis, migration and invasion, EMT, and weakening the cellular adhesion of ovarian cancer cells, and involved in the Bcl-2/Bax or the PI3K/Akt/mTOR pathway. Therefore, abnormal expression of GPR176 might be served as a biomarker for aggressive behaviour and poor prognosis of ovarian cancer and a target for gene therapy.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

CRISPR-Cas9 delivery strategies and applications: Review and update.

Nowadays, a significant part of the investigations carried out in the medical field belong to cancer treatment. Generally, conventional cancer treatments, including chemotherapy, radiotherapy, and surgery, which have been used for a long time, are not sufficient, especially in malignant cancers. Because genetic mutations cause cancers, researchers are trying to treat these diseases using genetic engineering tools. One of them is clustered regularly interspaced short palindromic repeats (CRISPR), a powerful tool in genetic engineering in the last decade. CRISPR, which forms the CRISPR-Cas structure with its endonuclease protein, Cas, is known as a part of the immune system (adaptive immunity) in bacteria and archaea. Among the types of Cas proteins, Cas9 endonuclease has been used in many scientific studies due to its high accuracy and efficiency. This review reviews the CRISPR system, focusing on the history, classification, delivery methods, applications, new generations, and challenges of CRISPR-Cas9 technology.

Development of an Improved Adenovirus Vector and Its Application to the Treatment of Lifestyle-Related Diseases.

The number of patients with lifestyle-related diseases such as type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), has continued to increase worldwide. Therefore, development of innovative therapeutic methods targeting lifestyle-related diseases is required. Gene therapy has attracted considerable attention as an advanced medical treatment. Safe and high-performance vectors are essential for the practical application of gene therapy. Replication-incompetent adenovirus (Ad) vectors are widely used in clinical gene therapy and basic research. Here, we developed a novel Ad vector, named Ad-E4-122aT, exhibiting higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. We also elucidated the mechanisms underlying Ad vector-induced hepatotoxicity during the early phase using Ad-E4-122aT. Next, we examined the therapeutic effects of the genes of interest, namely zinc finger AN1-type domain 3 (ZFAND3), lipoprotein lipase (LPL), and lysophospholipid acyltransferase 10 (LPLAT10), on lifestyle-related diseases using Ad-E4-122aT. We showed that the overexpression of ZFAND3 in the liver improved glucose tolerance and insulin resistance. Liver-specific LPL overexpression suppressed hepatic lipid accumulation and improved glucose metabolism. LPLAT10 overexpression in the liver suppressed postprandial hyperglycemia by increasing glucose-stimulated insulin secretion. Furthermore, we also focused on foods to advance research on the pathophysiology and treatment of lifestyle-related diseases. Cranberry and calamondin, which are promising functional foods, attenuated the progression of MASLD/NAFLD. Our findings will aid the development of new therapeutic methods, including gene therapy, for lifestyle-related diseases such as T2DM and MASLD/NAFLD.

CRISPR Advancements for Human Health.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful gene editing technology that is revolutionizing biomedical research and clinical medicine. The CRISPR system allows scientists to rewrite the genetic code in virtually any organism. This review provides a comprehensive overview of CRISPR and its clinical applications. We first introduce the CRISPR system and explain how it works as a gene editing tool. We then highlight current and potential clinical uses of CRISPR in areas such as genetic disorders, infectious diseases, cancer, and regenerative medicine. Challenges that need to be addressed for the successful translation of CRISPR to the clinic are also discussed. Overall, CRISPR holds great promise to advance precision medicine, but ongoing research is still required to optimize delivery, efficacy, and safety.

Emerging technology has a brilliant future: the CRISPR-Cas system for senescence, inflammation, and cartilage repair in osteoarthritis.

Osteoarthritis (OA), known as one of the most common types of aseptic inflammation of the musculoskeletal system, is characterized by chronic pain and whole-joint lesions. With cellular and molecular changes including senescence, inflammatory alterations, and subsequent cartilage defects, OA eventually leads to a series of adverse outcomes such as pain and disability. CRISPR-Cas-related technology has been proposed and explored as a gene therapy, offering potential gene-editing tools that are in the spotlight. Considering the genetic and multigene regulatory mechanisms of OA, we systematically review current studies on CRISPR-Cas technology for improving OA in terms of senescence, inflammation, and cartilage damage and summarize various strategies for delivering CRISPR products, hoping to provide a new perspective for the treatment of OA by taking advantage of CRISPR technology.

Comparative in vivo characterization of newly discovered myotropic adeno-associated vectors.

BACKGROUND: Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges, including a lack of transduction efficiency across the entire muscular system and toxicity resulting from off-target tissue effects. Recently, novel myotropic AAVs named MyoAAVs and AAVMYOs have been discovered using a directed evolution approach, all separately demonstrating enhanced muscle transduction efficiency and liver de-targeting effects. However, these newly discovered AAV variants have not yet been compared. METHODS: In this study, we performed a comparative analysis of these various AAV9-derived vectors under the same experimental conditions following different injection time points in two distinct mouse strains. RESULTS: We highlight differences in transduction efficiency between AAV9, AAVMYO, MyoAAV2A and MyoAAV4A that depend on age at injection, doses and mouse genetic background. In addition, specific AAV serotypes appeared more potent to transduce skeletal muscles including diaphragm and/or to de-target heart or liver. CONCLUSIONS: Our study provides guidance for researchers aiming to establish proof-of-concept approaches for preventive or curative perspectives in mouse models, to ultimately lead to future clinical trials for muscle disorders.

Engineered nanoparticles promote cardiac tropism of AAV vectors.

Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches.

An adeno-associated virus variant enabling efficient ocular-directed gene delivery across species.

Recombinant adeno-associated viruses (rAAVs) have emerged as promising gene therapy vectors due to their proven efficacy and safety in clinical applications. In non-human primates (NHPs), rAAVs are administered via suprachoroidal injection at a higher dose. However, high doses of rAAVs tend to increase additional safety risks. Here, we present a novel AAV capsid (AAVv128), which exhibits significantly enhanced transduction efficiency for photoreceptors and retinal pigment epithelial (RPE) cells, along with a broader distribution across the layers of retinal tissues in different animal models (mice, rabbits, and NHPs) following intraocular injection. Notably, the suprachoroidal delivery of AAVv128-anti-VEGF vector completely suppresses the Grade IV lesions in a laser-induced choroidal neovascularization (CNV) NHP model for neovascular age-related macular degeneration (nAMD). Furthermore, cryo-EM analysis at 2.1 Å resolution reveals that the critical residues of AAVv128 exhibit a more robust advantage in AAV binding, the nuclear uptake and endosome escaping. Collectively, our findings highlight the potential of AAVv128 as a next generation ocular gene therapy vector, particularly using the suprachoroidal delivery route.

In vivo genome editing via CRISPR/Cas9-mediated homology-independent targeted integration for Bietti crystalline corneoretinal dystrophy treatment.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.

The translational gap for gene therapies in low- and middle-income countries.

Gene therapies are designed to address the root cause of disease. As scientific understanding of disease prevention, diagnosis, and treatment improves in tandem with technological innovation, gene therapies have the potential to become safe and effective treatment options for a wide range of genetic and nongenetic diseases. However, as the medical scope of gene therapies expands, consideration must be given to those who will benefit and what proactive steps must be taken to widen development and access potential, particularly in regions carrying a high disease burden.