Adeno-associated viral vectors (AAVs) are promising tools for gene therapy. However, scaling up the production of AAVs to produce high-quality vectors at high yields for clinical purposes has proven to be challenging. In...Adeno-associated viral vectors (AAVs) are promising tools for gene therapy. However, scaling up the production of AAVs to produce high-quality vectors at high yields for clinical purposes has proven to be challenging. In the present study, we optimized the production process of AAV in a fixed-bed bioreactor using transient transfection in adherent HEK-293T cells. We systematically optimized the key process parameters, namely cell seeding density, cell density at transfection, and DNA-to-cell ratio, based on the yield obtained, starting from a prototype batch, followed by ten batch runs. Here, we packaged a reporter gene (enhanced green fluorescent protein) and a therapeutic gene (lysyl oxidase) into AAV9 capsids as part of our process development program to be applied for future current Good Manufacturing Practices production and clinical trial application. Throughout the experiments, media conditions, transfection processes, and mechanical parameters were kept identical, while monitoring pH, dissolved oxygen, and media glucose concentration during a production process of approximately 10 days. We demonstrate that by optimizing these parameters, the fixed-bed bioreactor was able to support as many as 1.6-2.8 × 10 cells/carrier strip, up to 3 × 10 cells/m bioreactor. Through this multivariate optimization process, we increased viral yield by about 7.6-fold (range of 5.7-10.4-fold for the optimized process runs) over the prototype batch. The total AAV vector yield average was 2.3 × 10 vg (range 1.1 × 10 vg to 4.95 × 10 vg), corresponding to an average per cell yield of 1.4 × 10 vg/cell (range 0.85 × 10-2.46 × 10vg/cell). In conclusion, our findings highlight that optimizing process parameters in a fixed-bed bioreactor presents a promising strategy for scalable and cost-effective AAV vector production.
Gene therapy has become a widely accepted treatment for inherited or acquired genetic diseases. Lentiviral vectors are of particular interest because of their favorable biosafety profile and ability to introduce their th...Gene therapy has become a widely accepted treatment for inherited or acquired genetic diseases. Lentiviral vectors are of particular interest because of their favorable biosafety profile and ability to introduce their therapeutic cargo into non-dividing cells. For clinical use, these viral vectors must be generated under conditions of good manufacturing practice in large quantities, which currently are provided via transient production. A solution for stable, robust, easy to scale, cost-effective, and predictable production of the therapeutic vectors is currently not available. Here, we describe the design, generation, and characterization of EL1-820, a packaging cell line for the stable production of lentiviral self-inactivating (SIN) vectors pseudotyped with the envelope glycoprotein of vesicular stomatitis virus. EL1-820 enables the introduction of a lentiviral SIN-vector expression cassette via Flp-recombinase-mediated cassette exchange (RMCE) into a predefined locus selected for optimal vector production, with expression units designed to improve reliability. EL1-820-based producer clones generated similar titers (1 × 10 TU/mL) from a targeted, single-copy integration of a lenti-GFP or a lenti-chimeric antigen receptor transfer vector as transient production. In initial scale-up experiments, multiple harvests from bioreactors could be achieved, resulting in titers of around 8-9 × 10 TU/mL after tangential flow filtration and a total yield of about 2.3 × 10 TU. In conclusion, RMCE-based introduction of the transfer construct allows stable, defined, predictable, and safe vector production suitable for clinical applications.
Recombinant adeno-associated virus (rAAV) vectors have emerged as a leading viral vector for gene therapy because they offer significant advantages over other viral vectors, such as stable physicochemical properties, lo...Recombinant adeno-associated virus (rAAV) vectors have emerged as a leading viral vector for gene therapy because they offer significant advantages over other viral vectors, such as stable physicochemical properties, low pathogenicity, low integration risk, and long-term expression of the transgene. In recent years, the number of rAAV products that have received approval for commercial marketing and clinical trials has grown rapidly, bringing hope for the treatment of refractory and rare diseases. However, rAAV products are highly innovative and complex, and the manufacturing processes to produce them are diverse and rapidly improving. Concurrently, the quality control methodologies and technologies are rapidly advancing and evolving. As biotechnology rapidly advances, there is a heightened need for communication between regulatory authorities and entities applying for rAAV products to be used for in-human trials or commercial marketing. Here, we focus on the discussion of chemistry, manufacturing, and control issues such as the control of adventitious viruses in different manufacturing processes and quality control during the biologics license application of rAAV products. It is expected to provide references and suggestions for the manufacturing and quality control of rAAV vectors, thereby accelerating the high-quality development of gene therapy products.
Dozens of gene therapies (GTs) have received regulatory approval, and hundreds more are in various research phases. To characterize the ethical, legal, and social implications (ELSI) associated with the clinical developm...Dozens of gene therapies (GTs) have received regulatory approval, and hundreds more are in various research phases. To characterize the ethical, legal, and social implications (ELSI) associated with the clinical development of this relatively new therapeutic class, we conducted a scoping review of the literature. Articles were eligible if they were written in English and discussed ELSI in the context of human somatic GT clinical research. 273 articles published from 2013 to 2024 met the eligibility criteria. To characterize and synthesize the ELSI associated with human somatic GT clinical research, thematic analysis was performed on extracted ELSI-relevant text from 179 articles published between 2019 and early 2024, including reviews, empirical research articles, opinions/commentaries, reports, news articles, and blog posts. Twenty-four themes were identified, leading to the generation of five high-level themes: (1) assessment of the risks and benefits of GTs is scientifically and ethically challenging, (2) communication and engagement with the patient community is crucial, (3) access and justice issues are heightened, (4) ethical GT trial design requires thoughtful consideration, and (5) strategic decision-making about GT research has ethical implications and is impacted by financial considerations and the regulatory context. Potential approaches to the identified ELSI are explored and discussed.
Myocardial fibrosis represents a maladaptive response to some pathological stimulus affecting the heart's functions, predisposing to arrhythmia and ultimately heart failure (HF). Stathmin 1 (Stmn1) is a core protein in r...Myocardial fibrosis represents a maladaptive response to some pathological stimulus affecting the heart's functions, predisposing to arrhythmia and ultimately heart failure (HF). Stathmin 1 (Stmn1) is a core protein in regulating the formation of the mitotic spindle. Although the dysregulation of Stmn1 has been confirmed to be related to the occurrence of kidney or liver fibrosis, the role of Stmn1 in HF remains obscure. In this study, bioinformatic analysis (GSE150736) of myocardial tissues from HF patients suggested that Stmn1 was significantly upregulated compared with healthy controls. The consistent results were also observed in the heart tissues of the rat model of HF. Furthermore, we demonstrated that Adenovirus-mediated overexpression of Stmn1 in the peri-infarct border zone area significantly improved cardiac fibrosis and collagen deposition, as evidenced by the decreased expression of transforming growth factor-beta1 (TGF-β1), collagen IV, and alpha-smooth muscle actin in heart tissues. , overexpression of Stmn1 reduced the production and deposition of collagen in cardiac fibroblasts (CFs) induced by TGF-β1, and thus inhibited the activation of CFs into myofibroblasts. Mechanistically, upregulation of Stmn1 significantly suppressed the phosphorylation of p38 both and . Moreover, we demonstrated that was transcriptionally regulated by the cohesin and CCCTC-binding factor () and functionally mediated the cardioprotective effects of Ctcf. Collectively, this work established Stmn1 as a promising therapeutic target for myocardial fibrosis in HF.
Baine S, Cui J, Seo YE
… +15 more, Kempton A, Rogers O, Haile A, Adegboye K, Wu J, Lemmerman L, Guerrero M, Jones C, Mayes H, Freehafer T, Lewis S, Pozsgai E, East L, Rodino-Klapac L, Potter R
Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is an ultra-rare autosomal recessive disorder caused by mutations in , the gene that encodes for β-sarcoglycan (SGCB), a component of the dystrophin-associated protei...Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is an ultra-rare autosomal recessive disorder caused by mutations in , the gene that encodes for β-sarcoglycan (SGCB), a component of the dystrophin-associated protein complex that stabilizes muscle fibers during contractions. Bidridistrogene xeboparvovec is an investigational adeno-associated virus-mediated gene transfer therapy designed to deliver a codon-optimized, full-length human and induce targeted expression of functional human SGCB protein. Interim safety and efficacy data from a clinical trial in patients with LGDM2E/R4 aged 4-15 years (NCT03652259) support further clinical development of bidridistrogene xeboparvovec. However, less is known about the effects of this agent in patients with more advanced LGMD2E/R4, who on average are older and heavier, which prompted their inclusion in studies VOYAGENE (NCT05876780, phase 1) and EMERGENE (NCT06246513, phase 3). In the preclinical study presented here, we delivered bidridistrogene xeboparvovec (0.185 × 10 vg/kg, 0.37 × 10 vg/kg, 0.74 × 10 vg/kg, 1.85 × 10 vg/kg, or 7.4 × 10 vg/kg) to -/- mice aged 27-42 weeks ( = 4 per dose) with age-matched saline-treated and C57BL/6J mice used as controls. Approximately 12 weeks after administration, we observed SGCB expression and found evidence of reduction in muscle fibrosis, reduction in muscle damage, and restoration of muscle force. Overall, a dose-dependent increase in vector exposure across tissue types was observed, with a nonlinear, exposure-dependent increase in both SGCB expression and functional improvement that reached saturation at 7.4 × 10 vg/kg. Pharmacokinetic and pharmacodynamic analyses demonstrated a robust relationship between vector biodistribution, SGCB expression, and muscle force, further supporting clinical development of bidridistrogene xeboparvovec at the highest dose (7.4 × 10 vg/kg), across a broad LGMD2E/R4 population and regardless of disease progression.
Recombinant adeno-associated virus (rAAV) vectors are among the most effective for gene therapy. A significant advancement in rAAV vector production is developing the triple-plasmid transfection method, which remains the...Recombinant adeno-associated virus (rAAV) vectors are among the most effective for gene therapy. A significant advancement in rAAV vector production is developing the triple-plasmid transfection method, which remains the most widely used technique. In this study, we used Expi293F (Expi293F) and Viral Production Cells 2.0 (VPC2.0 cells) to evaluate various transfection reagents, comparing transgene protein expression levels and intracellular plasmid copy numbers to optimize rAAV production. Our findings indicated that the effectiveness of transfection reagents in promoting higher rAAV production was cell-dependent and that rAAV productivity correlated more with plasmid levels in the cell nucleus than with transgene protein expression levels. Confocal laser microscopy revealed that in cells transfected with the high-yield transfection reagent, a large amount of free plasmid DNA entered the nucleus, whereas the transfection reagents themselves did not. These results provide new insights into the intracellular mechanisms underlying efficient rAAV vector production. Furthermore, identifying transfection reagents that facilitate nuclear plasmid delivery will aid in the selection of optimal reagents for high-yield AAV production.
Significant progress has been made in gene therapy for Duchenne muscular dystrophy (DMD), a severe genetic disorder primarily affecting pediatric patients. However, the immune responses triggered by high-dose systemic de...Significant progress has been made in gene therapy for Duchenne muscular dystrophy (DMD), a severe genetic disorder primarily affecting pediatric patients. However, the immune responses triggered by high-dose systemic delivery of adeno-associated virus (AAV) vectors remain a major challenge. These responses include the generation of long-lasting anti-capsid antibodies and potential immunity against the therapeutic transgene, rendering gene therapy ineffective. In addition, pre-existing anti-AAV antibodies exclude patients from eligibility for treatment. To address these limitations, we have developed an immunosuppression (IMS) strategy aimed at mitigating immune responses to the AAV capsid while enhancing microdystrophin expression. Using an optimized expression cassette (AAV9-UFµDys1) for sustained microdystrophin expression in striated muscle and heart, we observed a 40% improvement in muscle force compared with animals receiving a GFP-encoding control AAV9 vector. In mice, a single-dose IMS regimen significantly increased microdystrophin expression in cardiac and skeletal tissues and repeat dosing further enhanced expression, an effect not observed in non-IMS-treated mice. To model pre-existing immunity, we immune-challenged wild-type mice with empty AAV9 capsids and tracked antibody responses over time. The IMS regimen effectively reduced total anti-AAV antibody levels and increased microdystrophin expression in UFµDys1-treated mice. These findings highlight the potential of IMS to minimize immune barriers, facilitate repeat AAV administration, and expand the therapeutic window for DMD gene therapy. Our results support the further development of AAV-mediated approaches using either microdystrophin-expressing vectors or next-generation systems delivering full-length or near-full-length dystrophin.
Mutations in human , which encodes the collagen-like tail subunit (ColQ) of asymmetrical acetylcholinesterase (AChE), cause congenital myasthenic syndrome (CMS) with deficiency of end plate AChE. A valuable animal model...Mutations in human , which encodes the collagen-like tail subunit (ColQ) of asymmetrical acetylcholinesterase (AChE), cause congenital myasthenic syndrome (CMS) with deficiency of end plate AChE. A valuable animal model of -CMS is the Colq-deficient () mouse, which lacks asymmetrical AChE in skeletal and cardiac muscles. Mutant mice fail to thrive, and many die before reaching maturity. With the aim of developing a treatment for -CMS, mice were injected at postnatal day 26-29 with three doses of an adeno-associated virus type rh74 carrying full-length human (AAVrh74-): 5 × 10 viral genomes per kilogram (vg/kg) (intravenously [IV]), 1 × 10 vg/kg (IV), and 2 × 10 vg/kg (1 × 10 vg/kg IV + 1 × 10 vg/kg intraperitoneally). Motor performance was evaluated using rotarod, grip strength, and wire hang tests weekly for 12 weeks. Voluntary ambulation and repetitive nerve stimulation (RNS) were assessed once before euthanasia. Protein and RNA expression of COLQ was measured via immunohistochemistry (IHC) and reverse transcriptase quantitative PCR (RT-qPCR), respectively. Mice treated with AAVrh74- at 1 × 10 and 2 × 10 vg/kg doses showed 100% survival and no adverse side effects. Mice injected with 2 × 10 vg/kg showed almost full recovery and similar scores to wild type that were significantly higher than vehicle-injected mutants for grip strength ( value <0.0001), rotarod ( value <0.0001), and RNS ( value <0.0001). Similar improvements were observed in mice injected with 1 × 10 vg/kg, although the recovery of grip strength was incomplete. Mice injected with 5 × 10 vg/kg showed incomplete recovery. IHC demonstrated full recovery of protein expression in 1 × 10 and 2 × 10 vg/kg mice, and RT-qPCR unambiguously demonstrated that the source of the ColQ was human In summary, a single treatment of AAVrh74- (1 × 10 to 2 × 10 vg/kg) was effective and safe for mice, which reproduce many of the clinical features of the human -CMS phenotype. Thus, these results support a similar therapy for patients affected with -CMS.
Adeno-associated virus (AAV) vectors have emerged as the leading gene therapy vehicle due to their favorable safety profile and sustained payload expression. Approved therapies such as voretigene neparvovec (Luxturna) an...Adeno-associated virus (AAV) vectors have emerged as the leading gene therapy vehicle due to their favorable safety profile and sustained payload expression. Approved therapies such as voretigene neparvovec (Luxturna) and omnasemnogene abeparvovec (Zolgensma) rely on the tropism of natural AAV variants. The majority of discovered natural AAVs and engineered AAV capsids have not been comprehensively profiled for their biodistribution, especially at single-cell resolution. Recent advances in single nuclei sequencing can enable further refinement of AAV cell-type specificity and reduce off-target effects. However, low levels of transduction and muted sensitivity of current single-cell detection methods make screening pooled capsids at single-cell resolution challenging. Here, we develop SNAC (Single-Nuclei Atlas of Capsid distribution), an improved method for single-nuclei profiling of AAV transduction at multiplex scale. We provide proof of concept using the nonhuman primate eye as a model system, showing that we can accurately identify and quantify vector expression in all major retinal cell types. Furthermore, the ranking of capsids by SNAC agrees with that from pre-established tissue sampling protocols. Our method promises to reduce the time, effort, and cost of accurate cell-type-specific profiling of AAV capsids.
Iroanya GI, Subramanyam PN, Wells KD
… +1 more, Green JA
Hum Gene Ther
· 2025 Dec · PMID 40985164
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Adeno-associated virus (AAV) vectors have emerged as versatile and promising tools in gene therapy due to their favorable safety profile, broad tissue tropism, and long-term gene expression. However, pre-existing immunit...Adeno-associated virus (AAV) vectors have emerged as versatile and promising tools in gene therapy due to their favorable safety profile, broad tissue tropism, and long-term gene expression. However, pre-existing immunity, especially in the form of neutralizing antibodies (NAbs) remains a significant barrier, reducing vector efficacy and restricting patient eligibility. This review provides a comprehensive overview of the immunological landscape affecting AAV gene therapy, including global seroprevalence, environmental influences, and antibody cross-reactivity stemming from natural parvovirus exposure or vaccination of animal research models.We detail the mechanisms underlying immune detection and vector clearance, covering innate pattern recognition receptors, complement activation, and adaptive immune effector functions such as antibody-dependent complement deposition, cytotoxicity, and phagocytosis.We further analyze how species, age, serotype, administration route, and target tissue contribute to immune susceptibility and variable transduction outcomes. To overcome these challenges, we propose a three-pronged classification of mitigation strategies: (1) immune-focused strategies, such as plasmapheresis, immunoadsorption, enzymatic antibody cleavage, corticosteroids, and B cell depletion; (2) delivery-focused strategies, which include targeting immune-privileged sites, localized or intrathecal delivery, and timing of vector administration; and (3) capsid-focused strategies, comprising rational capsid engineering and the use of decoy particles or empty capsids.We also discuss promising advances such as AAV-specific regulatory T cells and re-dosable AAV platforms. This strategic framework offers a roadmap for tailoring gene therapy approaches to individual immune profiles and improving the safety, efficacy, and accessibility of AAVbased therapeutics.
Lonfat N, Moreno-Leon L, Punzo C
… +1 more, Khanna H
Hum Gene Ther
· 2025 Oct · PMID 40965251
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Inherited and complex retinal degenerative diseases, such as retinitis pigmentosa, age-related macular degeneration, and glaucoma, represent a significant global burden of irreversible vision loss. Due to immense genetic...Inherited and complex retinal degenerative diseases, such as retinitis pigmentosa, age-related macular degeneration, and glaucoma, represent a significant global burden of irreversible vision loss. Due to immense genetic and clinical heterogeneity and complex underlying mechanisms, these diseases still lack safe and effective disease-modifying treatments. This review summarizes the current landscape of gene therapeutic approaches to develop novel treatments for these blinding conditions. Specifically, we provide an update on several ongoing or completed clinical trials on gene-specific or gene-agnostic approaches, including recombinant adeno-associated viral vector-mediated delivery of the full gene or gene editing and antisense oligonucleotide components into the eye. We also discuss the initial clinical trial results of the use of the different approaches to ocular delivery, including subretinal, intravitreal, and suprachoroidal delivery. While long-term clinical trial data and refined clinical endpoints are essential to assess the efficacy, safety, and durability of these strategies, the data so far underscore the immense potential of gene therapy to revolutionize the management of retinal diseases in patients living with these debilitating conditions.
Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by defective ciliary motility, leading to recurrent respiratory infections and chronic airway damage. Gene therapy holds promise for treating PCD, but...Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by defective ciliary motility, leading to recurrent respiratory infections and chronic airway damage. Gene therapy holds promise for treating PCD, but its effectiveness in patient-derived models remains uncertain. This study aimed to evaluate the therapeutic potential of lentiviral gene delivery in restoring ciliary function in patient-derived nasal apical-out airway organoids. Using nasal epithelial cells from both healthy individuals and PCD patients with mutations in , , or , we established organoid models to assess gene therapy efficacy. Lentiviral vectors successfully restored the expression and proper localization of DNAAF proteins in mutant organoids, significantly improving ciliary beating frequency and the proportion of organoids with functional cilia. These findings provide proof-of-concept evidence supporting gene therapy as a viable approach to correct ciliary defects in PCD, paving the way for targeted treatments.
Adeno-associated virus (AAV)-based vectors are the most commonly used vectors for gene therapy. Wild-type AAV infections occur widely in humans and nonhuman primates (NHPs), and an accurate assessment of preexisting AAV...Adeno-associated virus (AAV)-based vectors are the most commonly used vectors for gene therapy. Wild-type AAV infections occur widely in humans and nonhuman primates (NHPs), and an accurate assessment of preexisting AAV antibodies is crucial for the efficient use of AAV-based gene therapies in preclinical and clinical studies. Cynomolgus macaques () are well-established preclinical large animal models for evaluating the efficacy and safety of AAV-mediated gene therapies intended for human use. We provide a retrospective evaluation comparing preexisting AAV-neutralizing or total antibody titers against serotypes AAV2, AAV5, AAV8, or AAV9 in cynomolgus macaque cohorts of Asian or Mauritian origin. We used an neutralizing antibody (NAB) assay to detect NAB titers or an Meso Scale Discovery-based assay for the quantification of total binding antibodies (TABs) in blood samples. Results were obtained to measure the serostatus of animals. In our analysis, the NAB assay revealed the lowest seroprevalence for AAV5 (13 ± 15% to 21 ± 6%) independent of origin. In the same assay, Asian animals were highly seropositive against AAV8, followed by AAV2 and AAV9 serotypes (88 ± 13%, 71 ± 10%, 69 ± 9%, respectively). Whereby, the prevalence of seropositivity was lower in animals of Mauritian origin with the highest seroprevalence for AAV9 (58 ± 7%), followed by AAV8 (53 ± 17%) and AAV2 (51 ± 20%) assessed by TAB assay. Notably, co-prevalences of antibody responses against AAV2, AAV8, and AAV9 serotypes resulted in 39.8% seropositivity ( NAB assay) in NHPs of Asian and in about 32.6% ( TAB assay) of Mauritian origin.
Gene therapy is emerging as a transformative approach for treating amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disease. While gene replacement has shown a groundbreaking success in spin...Gene therapy is emerging as a transformative approach for treating amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disease. While gene replacement has shown a groundbreaking success in spinal muscular atrophy, the complexity of ALS-due to frequent gain-of-function mutations and a heterogeneous etiology-presents significant challenges. Importantly, approximately 90% of ALS cases are sporadic, with unknown genetic mutation, further complicating patient stratification and therapeutic targeting. As a result, gene therapy strategies must often address multiple pathological mechanisms simultaneously. So far, current gene therapy strategies aim to either suppress toxic gene expression or promote neuroprotection, predominantly via viral-mediated delivery systems. This review will provide an overview of emerging preclinical and clinical gene therapy approaches for ALS, focusing on two main strategies: gene silencing and neuroprotection. Gene silencing techniques, including antisense oligonucleotides (ASOs), viral-mediated RNA interference, and gene editing, have demonstrated efficacy in reducing mutant gene expression, particularly in SOD1 and C9orf72 models, although clinical translation has so far yielded limited success. The recent Food and Drug Administration's approval of the ASO therapy Qalsody for SOD1-ALS underscores the clinical potential of these approaches. Neuroprotective strategies aim to enhance motor neuron survival through delivery of trophic factors, often targeting both central and peripheral tissues to harness retrograde transport mechanisms. We will discuss the advantages and limitations of various delivery vectors, targeting specificity, timing of intervention, and translational challenges, alongside current clinical trial data. This review aims to synthesize how these approaches may converge to address the multifaceted nature of ALS and guide the development of next-generation therapeutics.
Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considera...Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.