Gene Therapy For Diseases Research Articles

Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification

The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5′ end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5′ end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5′ end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production.

Lentiviral gene therapy for Fabry disease - 5 Year end of study analysis for the FACTS trial

Lentiviral gene therapy for Fabry disease - 5 Year end of study analysis for the FACTS trial

Use of the polymer PP6D5 and CRISPR-nCas9 in gene therapy for Tay-Sachs disease

Use of the polymer PP6D5 and CRISPR-nCas9 in gene therapy for Tay-Sachs disease

Correction of glycogen accumulation in muscle, heart and CNS in a pre-clinical model of hematopoietic stem cell gene therapy for Pompe disease

Correction of glycogen accumulation in muscle, heart and CNS in a pre-clinical model of hematopoietic stem cell gene therapy for Pompe disease

Pre-Clinical and Clinical Advances in Gene Therapy of X-Linked Retinitis Pigmentosa: Hope on the Horizon

X-linked retinitis pigmentosa (XLRP) is a severe inherited retinal degenerative disease characterized by progressive loss of photoreceptors and retinal pigment epithelium, leading to blindness. Predominantly affecting males due to mutations in the RPGR gene, XLRP currently lacks effective treatments beyond supportive care. Gene therapy has emerged as a promising approach to restore photoreceptor function by delivering functional copies of the RPGR gene. Recent clinical trials using AAV vectors, such as AAV5-RPGR and AGTC-501, have demonstrated encouraging results, including improvements in retinal sensitivity and visual function. While early successes like LUXTURNA have set the precedent for gene therapy in retinal diseases, adapting these strategies to XLRP presents unique challenges due to the complexity of RPGR mutations and the need for efficient photoreceptor targeting. Advances in vector design, including the use of optimized AAV serotypes with enhanced tropism for photoreceptors and specific promoters, have significantly improved gene delivery. Despite setbacks in some studies, ongoing research and clinical trials continue to refine these therapies, offering hope for patients affected by XLRP. This review explores the etiology and pathophysiology of XLRP, evaluates current treatment challenges, highlights recent clinical advances in gene therapy, and discusses future perspectives for bringing these therapies into clinical practice.

Lipid nanoparticle-mediated intracameral mRNA delivery facilitates gene expression and editing in the anterior chamber of the eye.

Lipid nanoparticles (LNPs) have shown great potential in the field of gene therapy for retinal diseases. To expand on this application, we investigated LNP-mediated mRNA delivery to the anterior chamber of the eye via the intracameral (IC) route of administration. Here, we show that IC injections of LNPs facilitated protein expression and gene editing in the trabecular meshwork (TM). Administration of Cre-mRNA LNPs to Ai9 mice resulted in robust tdTomato expression in the angle and corneal endothelium. In C57BL/6 mice, mCherry-mRNA LNPs demonstrated localized protein expression in the TM, which peaked at 72 h and subsequently declined over 120 h. Additionally, LNPs encapsulating Cas9 mRNA with sgAi9 enabled in vivo gene editing in Ai9 mice, with up to 14.3 % editing efficiency. This induced tdTomato expression in the iridocorneal angle, validating the potential of LNPs for gene editing applications. Importantly, no ocular toxicity was observed, indicating the safety of the IC LNP administration. Our findings highlight the use of LNPs for targeted gene therapy and editing, paving the path for the treatment of diseases such as glaucoma in the anterior eye.

Sickle cell disease gene therapy drug expenses and reimbursement: a litmus test for commercial pricing strategy and patient access for curative therapies.

Durable gene therapies newly approved by the Food and Drug Administration for sickle cell disease offer the promise of cure with subsequent improvement in quality of life through avoidance of repeated hospitalizations and worsening of disabilities in these patients. In the United States, following Food and Drug Administration approval, lovotibeglogene autotemcel (Lyfgenia, bluebird bio, Inc, Sommerville, MA, USA) and exagamglogene autotemcel (Casgevy, Vertex Pharmaceuticals Inc, Boston, MA, USA) were launched in 2024 for treatment of patients 12 years and older with frequent vaso-occlusive crises. Here we will discuss the first treatment with lovotibeglogene as a test case serving to highlight the multiple drug reimbursement pathways in the United States for cell and gene products for orphan disorders and their impact on patient adoption and access.

Screening of a retinal-targeting Adeno-Associated Virus (AAV) via DNA shuffling.

Due to its unique physiological structure and functions, the eye has received considerable attention in the field of adeno-associated virus (AAV) gene therapy. Inherited retinal degenerative diseases, which arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), are the most common cause of vision loss. However, current retinal gene therapy mostly involves subretinal injection of therapeutic genes, which treats a limited area, entails retinal detachment, and requires sophisticated techniques. Intravitreal (IVT) injection provides an alternative method with less invasion and more convenience for retinal gene therapy. In the present study, we performed a directed evolution via DNA shuffling in RHO-GFP mice and identified a novel recombinant AAV vector (AAV-M04) suitable for IVT injection in the gene delivery of retinal tissue. Compared with AAV2, AAV9, and AAV2.7m8, AAV-M04 vector exhibited higher transduction efficiency in retinal ganglion cell line-5 (RGC-5) cells as well as in human embryonic stem cell derived retinal organoids. Importantly, when delivered via IVT injection in mice, the AAV-M04 vector also showed better delivery efficiency of transgene as indicated by the red fluorescence protein mScarlet. The red fluorescence was distributed in a wider retinal area of AAV-M04 injected mice, suggesting the potent retinal targeting of AAV-M04 vector. In addition, AAV-M04 qualities including the packaging efficiency, the thermal stability, and the capsid integrity were superior to controls, which were important in drug manufacture. In summary, we screened a novel AAV-M04 vector with great retinal-targeting via IVT injection, which provides the potential of AAV-M04 for effective gene therapy of retinal diseases.

Advances in Gene Therapy for Rare Diseases: Targeting Functional Haploinsufficiency Through AAV and mRNA Approaches.

Most rare diseases (RDs) encompass a diverse group of inherited disorders that affect millions of people worldwide. A significant proportion of these diseases are driven by functional haploinsufficiency, which is caused by pathogenic genetic variants. Currently, most treatments for RDs are limited to symptom management, emphasizing the need for therapies that directly address genetic deficiencies. Recent advancements in gene therapy, particularly with adeno-associated viruses (AAVs) and lipid nanoparticle-encapsulated messenger RNA (mRNA), have introduced promising therapeutic approaches. AAV vectors offer durable gene expression, extensive tissue tropism, and a safety profile that makes them a leading choice for gene delivery; however, limitations remain, including packaging size and immune response. In contrast, mRNA therapeutics, formulated in LNPs, facilitate transient protein expression without the risk of genomic integration, supporting repeated dosing and pharmacokinetic control, though with less long-term expression than AAVs. This review analyzes the latest developments in AAV and mRNA technologies for rare monogenic disorders, focusing on preclinical and clinical outcomes, vector design, and delivery challenges. We also address key regulatory and immunological considerations impacting therapeutic success. Together, these advancements in AAV and mRNA technology underscore a new era in RD treatment, providing innovative tools to target the genetic root of these diseases and expanding therapeutic approaches for patients who currently face limited medical options.

A Smart mRNA-Initiated Theranostic Multi-shRNA Nanofactory for Precise and Efficient Cancer Gene Therapy.

Despite the significant potential of short hairpin RNA (shRNA)-mediated gene therapy for various diseases, the clinical success of cancer treatment remains poor, partly because of low selectivity and low efficiency. In this study, an mRNA-initiated autonomous multi-shRNA nanofactory (RNF@CM) is designed for in vivo amplification imaging and precise cancer treatment. The RNF@CM consists of a gold nanoparticle core, an interlayer of two types of three-stranded DNA/RNA hybrid probes, one of which is bound to aptamer-inhibited DNA polymerases, and an outer layer of the cancer cell membrane. After the specific delivery of RNF@CM into target cancer cells, an intracellular tumour-related mRNA target can initiate the RNF@CM with a circular strand-displacement polymerisation reaction, resulting in the release of significantly amplified fluorescence and continuous production of three types of shRNAs. The RNF@CM effectively distinguished cancer cells from normal cells, exclusively produced multiple shRNAs in response to a specific mRNA target in cancer cells, accurately diagnosed tumours in vivo, and significantly inhibited tumour growth with negligible toxicity, expanding the toolbox for on-demand gene delivery and precision theranostics.

Lentivirus-mediated gene therapy for Fabry disease: 5-year End-of-Study results from the Canadian FACTs trial.

Fabry disease is an X-linked lysosomal storage disorder due to a deficiency of α-galactosidase A (α-gal A) activity. Our goal was to correct the enzyme deficiency in Fabry patients by transferring the cDNA for α-gal A into their CD34+ hematopoietic stem/progenitor cells (HSPCs). Overexpression of α-gal A leads to secretion of the hydrolase; which can be taken up and used by uncorrected bystander cells. Gene-augmented HSPCs can circulate and thus provide sustained systemic correction. Interim results from this 'first-in-the-world' Canadian FACTs (Fabry Disease Clinical Research and Therapeutics) trial were published in 2021. Herein we report 5-year 'End-of-Study' results. Five males with classical Fabry disease were treated. Their HSPCs were mobilized, enriched, and transduced with a recombinant lentivirus engineering expression of α-gal A. Autologous transduced cells were infused after conditioning with a nonmyeloablative, reduced dose, melphalan regimen. Safety monitoring was performed. α-Gal A activity was measured in plasma and peripheral blood (PB) leucocytes. Globotriaosylceramide (Gb3) and lyso-Gb3 levels in urine and plasma were assessed by mass spectrometry. qPCR assays measured vector copy number in PB leucocytes. Antibody titers were measured by ELISA. Body weight, blood pressure, urinary protein levels, eGFR, troponin levels, and LVMI were tracked. Four out of 5 patients went home the same day as their infusions; one was kept overnight for observation. Circulating α-gal A activity was observed at Day 6-8 in each patient following infusion and has remained durable for 5+ years. LV marking of peripheral blood cells has remained durable and polyclonal. All 5 patients were eligible to come off biweekly enzyme therapy; 3 patients did so. Plasma lyso-Gb3 was significantly lower in 4 of 5 patients. There was no sustained elevation of anti-α-gal A antibodies. Patient weight was stable in 4 of the 5 patients. All blood pressures were in the normal range. Kidney symptoms were stabilized in all patients. This treatment was well tolerated as only two SAEs occurred (during the treatment phase) and only two AEs were reported since 2021. We demonstrate that this therapeutic approach has merit, is durable, and should be explored in a larger clinical trial. This was the first gene therapy clinical trial to be completed for Fabry disease. There were no adverse events of any grade attributable to the cellular gene therapy intervention or host conditioning throughout the follow-up interval of 5 years. After reduced-intensity melphalan treatment, all patients engrafted their autologous modified α-gal A expressing cells. All patients synthesized and secreted α-gal A throughout the course of the study. Expression of α-gal A resulted in a decrease in plasma lyso-Gb3 in four of five patients and stabilization of kidney symptoms in all patients.

Development of an in vitro model of dysferlinopathy via crispr/cas-mediated transcriptional activation of the dysf gene

Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening in order to select the most effective approach to restore the synthesis of the target protein. We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.

CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.

In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.

Adenoviral Vectors for Gene Therapy of Hereditary Diseases.

Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, high capacity, and high transduction efficiency, which makes them actively used as oncolytic vectors and for creating vector vaccines. However, despite their numerous advantages, AdVs have not yet found their place in gene therapy for hereditary diseases. This review provides an overview of AdVs, their features, and clinical trials using them for gene replacement therapy in monogenic diseases and analyzes the reasons for the failures of these studies. Additionally, current research on the modification of AdVs to reduce immune responses and target delivery is discussed.

Gene therapy in polycystic kidney disease: A promising future.

Polycystic kidney disease (PKD) is a genetic disorder marked by numerous cysts in the kidneys, progressively impairing renal function. It is classified into autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), with ADPKD being more common. Current treatments mainly focus on symptom relief and slowing disease progression, without offering a cure. Recent advancements in gene editing technologies, such as CRISPR-Cas9, have introduced new therapeutic possibilities for PKD. These approaches include miR-17 antisense oligonucleotides, adenovirus-mediated gene knockdown, Pkd1 gene or polycystin -1 C-terminal tail enhancement therapy, and 3-UTR miR-17 binding element by CRISPR-Cas9, which have shown potential in animal models and early clinical trials. Specifically for ARPKD, strategies like antisense oligonucleotide therapy targeting c-myc and CRISPR/ Cas9 knockdown of the P2rx7 gene have shown promise. Despite facing challenges such as technological limitations, ethical and legal issues, and high costs, gene therapy presents unprecedented hope for PKD treatment. Future interdisciplinary collaboration and international cooperation are essential for developing more effective treatment strategies for PKD patients.

Evolving Concept of Value in Health Economics and Outcomes Research: Emerging Tools for Innovation and Access to Cell and Gene Therapies for Rare Diseases

Evolving Concept of Value in Health Economics and Outcomes Research: Emerging Tools for Innovation and Access to Cell and Gene Therapies for Rare Diseases

Focused Ultrasounds as an Adeno-Associated Virus Gene Therapy-Empowering Tool in Juvenile Mice via Intracerebroventricular Administration.

Systemic delivery of adeno-associated virus (AAV) vectors targeting the central nervous system has the potential to solve many neurodevelopmental disorders, yet it is made difficult by the filtering effect of the blood-brain barrier and systemic complications. To overcome this limitation, we attempted to inject a Venus-expressing, oligodendrocyte-selective AAV9 viral vector in the ventricles together with lipid microbubbles and subjected them to focused ultrasound (FUS); the resulting mechanical stimulation on the brain ventricles is able to open small, temporary gaps from which vector particles can leak and spread. Our findings indicate that FUS can increase viral vector diffusion across both the anteroposterior and left-right axes without influencing cell tropism; significant effects were found with 60 and 90 s exposure time, but no effects were observed with longer intervals. Taken together, these results highlight a new strategy for the safe and effective delivery of viral vectors and offer new perspectives for the development and application of gene therapies for central nervous system diseases.

State of the art and perspectives of gene therapy in heart failure. A scientific statement of the Heart Failure Association of the ESC, the ESC Council on Cardiovascular Genomics and the ESC Working Group on Myocardial & Pericardial Diseases.

Gene therapy has recently become a reality in the treatment of cardiovascular diseases. Strategies to modulate gene expression using antisense oligonucleotides or small interfering RNA are proving to be safe and effective in the clinic. Adeno-associated viral vector-based gene delivery and CRISPR-Cas9-based genome editing have emerged as efficient strategies for gene delivery and repair in humans. Overall, gene therapy holds the promise not only of expanding current treatment options, but also of intervening in previously untackled causal disease mechanisms with little side effects. This scientific statement provides a comprehensive overview of the various modalities of gene therapy used to treat heart failure and some of its risk factors, and their application in the clinical setting. It discusses specifically the possibilities of gene therapy for hereditary heart diseases and (non)-genetic heart failure. Furthermore, it addresses safety and clinical trial design issues and challenges for future regulatory strategies.

Advances and Challenges in Gene Therapy for Neurodegenerative Diseases: A Systematic Review.

This review explores recent advancements in gene therapy as a potential treatment for neurodegenerative diseases, focusing on intervention mechanisms, administration routes, and associated limitations. Following the PRISMA procedure guidelines, we systematically analyzed studies published since 2020 using the PICO framework to derive reliable conclusions. The efficacy of various gene therapies was evaluated for Parkinson's disease (n = 12), spinal muscular atrophy (n = 8), Huntington's disease (n = 3), Alzheimer's disease (n = 3), and amyotrophic lateral sclerosis (n = 6). For each condition, we assessed the therapeutic approach, curative or disease-modifying potential, delivery methods, advantages, drawbacks, and side effects. Results indicate that gene therapies targeting specific genes are particularly effective in monogenic disorders, with promising clinical outcomes expected in the near future. In contrast, in polygenic diseases, therapies primarily aim to promote cell survival. A major challenge remains: the translation of animal model success to human clinical application. Additionally, while intracerebral delivery methods enhance therapeutic efficacy, they are highly invasive. Despite these hurdles, gene therapy represents a promising frontier in the treatment of neurodegenerative diseases, underscoring the need for continued research to refine and personalize treatments for each condition.

Gene Therapy for Parkinson's Disease Using Midbrain Developmental Genes to Regulate Dopaminergic Neuronal Maintenance.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder. It is characterized by the progressive loss of dopaminergic (DAnergic) neurons in the substantia nigra and decreased dopamine (DA) levels, which lead to both motor and non-motor symptoms. Conventional PD treatments aim to alleviate symptoms, but do not delay disease progression. PD gene therapy offers a promising approach to improving current treatments, with the potential to alleviate significant PD symptoms and cause fewer adverse effects than conventional therapies. DA replacement approaches and DA enzyme expression do not slow disease progression. However, DA replacement gene therapies, such as adeno-associated virus (AAV)-glutamic acid decarboxylase (GAD) and L-amino acid decarboxylase (AADC) gene therapies, which increase DA transmitter levels, have been demonstrated to be safe and efficient in early-phase clinical trials. Disease-modifying strategies, which aim to slow disease progression, appear to be potent. These include therapies targeting downstream pathways, neurotrophic factors, and midbrain DAnergic neuronal factors, all of which have shown potential in preclinical and clinical trials. These approaches focus on maintaining the integrity of DAnergic neurons, not just targeting the DA transmitter level itself. In particular, critical midbrain developmental and maintenance factors, such as Nurr1 and Foxa2, can interact synergistically with neighboring glia, in a paracrine mode of action, to protect DAnergic neurons against various toxic factors. Similar outcomes could be achieved by targeting both DAnergic neurons and glial cells with other candidate gene therapies, but in-depth research is needed. Neurotrophic factors, such as neurturin, the glial-cell-line-derived neurotrophic factor (GDNF), the brain-derived neurotrophic factor (BDNF), and the vascular endothelial growth factor (VEGF), are also being investigated for their potential to support DAnergic neuron survival. Additionally, gene therapies targeting key downstream pathways, such as the autophagy-lysosome pathway, mitochondrial function, and endoplasmic reticulum (ER) stress, offer promising avenues. Gene editing and delivery techniques continue to evolve, presenting new opportunities to develop effective gene therapies for PD.