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[Display omitted] • The coexistence of mutant and wild-type mtDNA constitutes the heteroplasmy of mtDNA. • The ovary is a highly energy-intensive organ, and it relies heavily on mitochondrial function. • Mitochondrial heteroplasmy can potentially contribute to a variety of significant ovarian disorders. • Current strategies related to mitochondrial heteroplasmy are untargeted and have low bioavailability. • The exploration of new mitochondria-targeted therapeutic strategies may offer promising paths towards these diseases. The reportedly high mutation rate of mitochondrial DNA (mtDNA) may be attributed to the absence of histone protection and complete repair mechanisms. Mitochondrial heteroplasmy refers to the coexistence of wild-type and mutant mtDNA. Most healthy individuals carry a low point mutation load (<1 %) in their mtDNA, typically without any discernible phenotypic effects. However, as it exceeds a certain threshold, it may cause the onset of various diseases. Since the ovary is a highly energy-intensive organ, it relies heavily on mitochondrial function. Mitochondrial heteroplasmy can potentially contribute to a variety of significant ovarian disorders. In this review, we have elucidated the close relationship between mtDNA heteroplasmy and ovarian diseases, and summarized novel avenues and strategies for the potential treatment of these ovarian diseases. Mitochondrial heteroplasmy can potentially contribute to a variety of significant ovarian disorders, including polycystic ovary syndrome, premature ovarian insufficiency, and endometriosis. Current strategies related to mitochondrial heteroplasmy are untargeted and have low bioavailability. Nanoparticle delivery systems loaded with mitochondrial modulators, mitochondrial replacement/transplantation therapy, and mitochondria-targeted gene editing therapy may offer promising paths towards potentially more effective treatments for these diseases, despite ongoing challenges. [ABSTRACT FROM AUTHOR]

Genetic manipulation of hematopoietic stem cells (HSCs) is being developed as a therapeutic strategy for several inherited disorders. This field is rapidly evolving with several novel tools and techniques being employed to achieve desired genetic changes. While commercial products are now available for sickle cell disease, transfusion-dependent β-thalassemia, metachromatic leukodystrophy and adrenoleukodystrophy, several challenges remain in patient selection, HSC mobilization and collection, genetic manipulation of stem cells, conditioning, hematologic recovery and post-transplant complications, financial issues, equity of access and institutional and global preparedness. In this report, we explore the current state of development of these therapies and provide a comprehensive assessment of the challenges these therapies face as well as potential solutions. [ABSTRACT FROM AUTHOR]

Ophthalmic treatment demands precision and consistency in delivering therapeutic agents over extended periods to address many conditions, from common eye disorders to complex diseases. This diversity necessitates a range of delivery strategies, each tailored to specific needs. We delve into various delivery cargos that are pivotal in ophthalmic care. These cargos encompass biodegradable implants that gradually release medication, nonbiodegradable implants for sustained drug delivery, refillable tools allowing flexibility in treatment, hydrogels capable of retaining substances while maintaining ocular comfort, and advanced nanotechnology devices that precisely target eye tissues. Within each cargo category, we explore cutting-edge research-level approaches and FDA-approved methods, providing a thorough overview of the current state of ophthalmic drug delivery. In particular, our focus on nanotechnology reveals the promising potential for gene delivery, cell therapy administration, and the implantation of active devices directly into the retina. These advancements hold the key to more effective, personalized, and minimally- invasive ophthalmic treatments, revolutionizing the field of eye care. [ABSTRACT FROM AUTHOR]

Liver fibrosis is characterized by abnormal accumulation of extracellular matrix proteins, disrupting normal liver function. Despite its significant health impact, effective treatments remain limited. Here, we present the development of engineered lipid nanoparticles (LNPs) for targeted RNA therapeutic delivery in the liver. We investigated the therapeutic potential of modulating the G2 and S-phase expressed 1 (GTSE1) protein for treating liver fibrosis. Through screening, we identified P13 8Y LNP as a potent candidate with superior delivery efficiency and lower toxicity. Using these engineered LNPs, we successfully delivered siGTSE1 to hepatocytes, significantly reducing collagen accumulation and restoring liver function in a fibrosis animal model. Additionally, GTSE1 downregulation altered miRNA expression and upregulated hepatocyte nuclear factor 4 alpha (HNF4α). These findings suggest that therapeutic gene silencing of GTSE1 is a promising strategy for treating liver fibrosis by regenerating liver phenotypes and functions. A significant decrease in GTSE1 levels, achieved by targeted delivery of siGTSE1 using engineered LNPs, leads to reduced collagen deposition and restoration of liver function through alterations in liver fibrosis-related miRNA expression and the upregulation of hepatocyte nuclear factor 4 alpha (HNF4⍺). [Display omitted] [ABSTRACT FROM AUTHOR]

Self-amplifying RNA (saRNA) is a next-generation RNA platform derived from an alphavirus that enables replication in host cytosol, offering a promising shift from traditional messenger RNA (mRNA) therapies by enabling sustained protein production from minimal dosages. The approval of saRNA-based vaccines, such as the ARCT-154 for COVID-19 in Japan, underscores its potential for diverse therapeutic applications, including vaccine development, cancer immunotherapy, and gene therapy. This study investigates the role of delivery vehicle and administration route on saRNA expression kinetics and reactogenicity. Employing ionizable lipid-based nanoparticles (LNPs) and polymeric nanoparticles, we administered saRNA encoding firefly luciferase to BALB/c mice through six routes (intramuscular (IM), intradermal (ID), intraperitoneal (IP), intranasal (IN), intravenous (IV), and subcutaneous (SC)), and observed persistent saRNA expression over a month. Our findings reveal that while LNPs enable broad route applicability and stability, pABOL (poly (cystamine bisacrylamide- co -4-amino-1-butanol)) formulations significantly amplify protein expression via intramuscular delivery. Notably, the disparity between RNA biodistribution and protein expression highlight the nuanced interplay between administration routes, delivery vehicles, and therapeutic outcomes. Additionally, our research unveiled distinct biodistribution profiles and inflammatory responses contingent upon the chosen delivery formulation and route. This research illuminates the intricate dynamics governing saRNA delivery, biodistribution and reactogenicity, offering essential insights for optimizing therapeutic strategies and advancing the clinical and commercial viability of saRNA technologies. [Display omitted] • Intraperitoneal route enables broader biodistribution and higher saRNA expression. • LNPs excel in RNA delivery efficiency but induce significant inflammation. • Higher RNA transfection does not always correlate with higher protein expression. • Reactogenicity is modulated by biodistribution. [ABSTRACT FROM AUTHOR]

Hirschsprung's disease (HD) is a congenital functional disorder characterized by the absence of ganglion cells in the enteric nervous system. The estimated incidence of HD is 1 in 5000 live births with up to 60% of patients having associated anomalies. This short article aims to provide paediatricians with an overview of the condition and the recent developments in our understanding. Short-segment rectosigmoid disease is seen in 75% of cases with infants presenting with abdominal distension, bilious vomiting and delayed passage of meconium. A bedside rectal suction biopsy is the gold-standard in confirming the diagnosis of HD. The absence of ganglion cells in the myenteric and submucosal plexi in the presence of thickened hypertrophic nerves (more than40 microns diameter) is diagnostic of HD. There are no medical options to treat HD, however rectal washouts can be used to bridge the gap to surgery. Contemporary management of this condition involves a single-stage pull-through that can be performed using various techniques including minimal access surgery. Long-term problems in with HD are common and these include obstructive symptoms (10–30%), constipation (10–15%), enterocolitis (10–15%) and true faecal incontinence (1–2%). Furthermore 20–30% of adults report symptoms of incontinence and constipation as well as poor sexual function. Future research is being focussed on novel gene therapies including stem-cell therapy and the use of CRISPR/CA9 to edit parts of the genome. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10–40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs. • FectoVIR significantly increased rAAV9 potency compared to other reagents. • About 80 % of rAAV capsids are fully filled, with no difference due to reagent choice, as shown by AUC and mass photometry. • FectoVIR improves rAAV9 genome integrity, as shown by results from multiplex dPCR and Nanopore sequencing. • Preparation steps preceding Nanopore sequencing affect the distribution of fragments and the accuracy of titer measurements. • Employing a combination of mass- and sequence-based analytical methods is crucial for the thorough characterization of rAAV9s. [ABSTRACT FROM AUTHOR]

Local gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. To optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. AAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. A novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. The combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies. • A novel AAV capsid termed AAVDJK2 was developed and was superior to existing AAVs in terms of gene transduction efficiency and specificity for keratinocytes in vitro and in vivo. • A novel tissue-specific promotor termed K14 SCP3 promotor was developed and was superior to existing promoters in terms of gene transduction efficiency and specificity for keratinocytes in vitro and in vivo. • The novel AAV system benefits experimental research and development of new epidermis-targeted gene therapies. [ABSTRACT FROM AUTHOR]

In an earlier investigation, our group introduced the TFAMoplex, a transfection agent based on the mitochondrial transcription factor A (TFAM) protein, which complexes DNA into nanoparticles. The original TFAMoplex further contained a bacterial phospholipase to achieve endosomal escape, and the vaccinia-related kinase 1 (VRK1), which significantly boosted the transfection efficiency of the system by an unknown mechanism. This study aims at replacing VRK1 within the TFAMoplex with dynein light chain proteins, specifically RP3, to directly tether the complexes to the dynein motor complex for enhanced cytosolic transport. To confirm the interaction between the dynein complex and the resulting fusion protein, we examined the binding kinetics of TFAM-RP3 to the dynein intermediate chains 1 and 2. Furthermore, we established a proteomics-based assay to compare cytosolic protein interactions of different TFAMoplex variants, including the RP3-modified version and the original VRK1-containing system. In the group of the VRK1-containing TFAMoplex, significant shifts of protein interactors were observed, especially for nucleolar proteins. Leveraging this knowledge, we incorporated one of these nuclear proteins, leucine-rich repeat-containing protein 59 (LRRC59), into the TFAMoplex, resulting in a significant improvement of transfection properties compared to the RP3-modified system and comparable levels versus the original, VRK1-containing version. This study not only advances our comprehension of the TFAMoplex system but also offers broader insights into the potential of protein engineering for designing effective gene delivery systems. [Display omitted] [ABSTRACT FROM AUTHOR]

Lipid-based complex injectables are renowned for their effectiveness in delivering drugs, with many approved products. While significant strides have been made in formulating nanosystems for small molecular weight drugs, a pivotal breakthrough emerged with the recognition of lipid nanoparticles as a promising platform for delivering nucleic acids. This finding has paved the way for tackling long-standing challenges in molecular and delivery aspects (e.g., mRNA stability, intracellular delivery) that have impeded the clinical translation of gene therapy, especially in the realm of immunotherapy. Nonetheless, developing and implementing new lipid-based delivery systems pose significant challenges, as industrial manufacturing of these formulations often involves complex, multi-batch processes, giving rise to issues related to scalability, stability, sterility, and regulatory compliance. To overcome these obstacles, embracing the principles of quality-by-design (QbD) is imperative. Furthermore, adopting cutting-edge manufacturing and process analytical tools (PAT) that facilitate the transition from batch to continuous production is essential. Herein, the key milestones and insights derived from the development of currently approved lipid- nanosystems will be explored. Additionally, a comprehensive and critical overview of the latest technologies and regulatory guidelines that underpin the creation of more efficient, scalable, and flexible manufacturing processes for complex lipid-based nanoformulations will be provided. [Display omitted] • Lipid-based complex injectables reliably deliver drugs with many approved products. • Lipid nanoparticles significantly enhance nucleic acid delivery for gene therapy. • Multi-batch processes challenge scalability, stability, and compliance. • Quality-by-Design ensures consistent product quality in complex manufacturing. • Process Analytical Tools enhance efficiency and flexibility in manufacturing. [ABSTRACT FROM AUTHOR]

Subretinal injection is the preferred delivery technique for various novel ocular therapies and is widely used because of its precision and efficient delivery of gene and cell therapies; however, choosing an injection point and defining delivery parameters to target a specified retinal location and area is an inexact science. We provide an overview of the key factors that play important roles during subretinal injections to refine the technique, enhance patient outcomes, and minimise risks. We describe the role of anatomical and physical variables that affect subretinal bleb propagation and shape and their impact on retinal integrity. We highlight the risks associated with subretinal injections and consider strategies to mitigate reflux and retinal trauma. Finally, we explore the emerging field of robotic assistance in improving intraocular manouvrability and precision to facilitate the injection procedure. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss among the elderly in Western communities, with an estimated global prevalence of 10 – 20% in people older than 65 years. AMD leads to central vision loss due to degeneration of the photoreceptors, retinal pigment epithelium and the choriocapillaris. Beckman's classification for AMD, based upon color fundus photographs, divides the disease into early, intermediate, and late forms. The late, vision-threatening stage includes both neovascular AMD and geographic atrophy. Despite its high prevalence and impact on patients' quality of life, treatment options for AMD are limited. While neovascular AMD can be medically managed with anti-VEGF intravitreal injections, until very recently there has been no approved treatment options for atrophic AMD; however, in February 2023 the first treatment for geographic atrophy – pegcetacoplan – was approved by the US FDA. We describe the current landscape of potential gene and cell therapeutic strategies for late-stage AMD, with an emphasis on the therapeutic options that might become available in the next few years. [ABSTRACT FROM AUTHOR]

• Heaviest treated patients in the real world with onasemnogene abeparvovec. • Liver impairment and only transitory improvement in functional outcomes. • Risk-benefit considerations is required in treating older and heavier SMA patients. Spinal muscular atrophy (SMA) is an autosomal recessive disorder with progressive muscle atrophy and weakness, caused by biallelic mutations in the survival motor neuron 1 (SNM1) gene. Onasemnogene abeparvovec (OA) is an approved gene replacement therapy for patients with SMA. We report on two patients with SMA type 1, weighing 20 kg, previously treated with Nusinersen, who received OA infusion at 7 years of age. To our knowledge, these two patients are the heaviest treated in the real-world and we describe their different courses after gene therapy, including liver impairment requiring long-term steroid treatment and additional immunosuppression, with only transitory improvement in functional outcomes. Our cases illustrate how careful risk-benefit consideration is required in treating older and heavier SMA patients with OA, especially in view of the multiple treatment choices available for older patients with SMA. [ABSTRACT FROM AUTHOR]

The use of recombinant adeno-associated virus (AAV) vectors is a popular choice for in vivo gene therapy, with hundreds of ongoing clinical trials targeting various genetic diseases. However, due to limited material availability and the complexity of AAV structure, there is a critical lack of comprehensive studies on AAV degradation pathways. In this study, we intended to elucidate the degradation pathways for a model AAV9 with GFP as the transgene under relevant stressed conditions. We assessed a diverse set of critical quality attributes and examined the overall impact of various stresses on transgene expression. This assessment revealed various degradation mechanisms of AAV9 and demonstrated the potential risk of a base formulation in causing AAV9 instability and potency loss under thermal stress at 25 and 40 °C while maintaining stability under freeze-thaw stress, interfacial stress due to membrane filtration, and short-term storage of up to 4 weeks at 5 °C. [ABSTRACT FROM AUTHOR]

Cotoretigene toliparvovec (BIIB112/AAV8-RPGR) is an investigational vector-based gene therapy designed to provide a full-length, codon-optimized retinitis pigmentosa GTPase regulator (RPGR) protein to individuals with RPGR -associated X-linked retinitis pigmentosa (XLRP). We assessed efficacy and tolerability of cotoretigene toliparvovec subretinal gene therapy. Part 2 of the XIRIUS trial (ClinicalTrials.gov identifier, NCT03116113) was a phase 2/3, 12-month, randomized (1:1:1) dose-expansion study. Male patients ≥10 years of age with RPGR -associated XLRP were included. Participants were randomized 1:1:1 to receive low-dose subretinal cotoretigene toliparvovec (5 × 1010 vector genomes/eye), high-dose cotoretigene toliparvovec (2.5 × 1011 vector genomes/eye) or to be an untreated control participant. The primary end point was the percentage of participants meeting microperimetry responder criteria (≥ 7-dB improvement at ≥ 5 of 16 central loci). Secondary end points included change from baseline in retinal sensitivity at the central 16 loci and the entire 68 loci at 12 months and change from baseline in low-luminance visual acuity (LLVA) at 12 months, as well as the proportion of eyes with a ≥ 15-Early Treatment Diabetic Retinopathy Study ETDRS letter LLVA and ≥ 10-ETDRS letter LLVA change from baseline at month 12. Because of the impact of the COVID-19 pandemic, enrollment ended before reaching the initial target, leaving the trial underpowered. Twenty-nine participants were included (low-dose group, n = 10; high-dose group, n = 10; control group, n = 9). At month 12, the percentage of participants meeting microperimetry responder criteria was not significantly different between either cotoretigene toliparvovec group (low dose, 37.5% [ P = 0.3181]; high dose, 25.0% [ P = 0.5177]) and the control group (22.2%). However, the mean change from baseline in microperimetry sensitivity improved significantly with the low-dose group versus the control group at month 12 (P = 0.0350). Significant improvement in LLVA occurred in the low-dose group versus the control group at month 12 (33.3% difference [80% confidence interval, 14.7%–55.2%]; P = 0.0498). Three ocular-related serious adverse events (SAEs) occurred in the low-dose group versus 7 SAEs in the high-dose group. The primary microperimetry end point was not met. Significant improvements in LLVA and mean microperimetry were observed compared with controls and fewer SAEs occurred with low-dose compared with high dose cotoretigene toliparvovec. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. [ABSTRACT FROM AUTHOR]

[Display omitted] • Alox5 deficiency in neuron alleviates seizure activity and epileptogenesis. • Neuronal Alox5 deletion reverses neuron loss and astrogliosis. • The development of comorbidities in the chronic phase of epilepsy was alleviated by Alox5 deletion. • Anti-epileptic effect of neuronal Alox5 deletion may be linked with reducing glutamate. • The identification of Alox5 as a potential therapeutic target suggests its promising role in the management of epilepsy. Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn ISer603. Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy. [ABSTRACT FROM AUTHOR]

To illustrate the financial consequences of implementing different managed entry agreements (managed entry agreements for the Dutch healthcare system for autologous gene therapy atidarsagene autotemcel [Libmeldy]), while also providing a first systematic guidance on how to construct managed entry agreements to aid future reimbursement decision making and create patient access to high-cost, one-off potentially curative therapies. Three payment models were compared: (1) an arbitrary 60% price discount, (2) an outcome-based spread payment with discounts, and (3) an outcome-based spread payment linked to a willingness to pay model with discounts. Financial consequences were estimated for full responders (A), patients responding according to the predicted clinical pathway presented in health technology assessment reports (B), and unstable responders (C). The associated costs for an average patient during the time frame of the payment agreement, the total budget impact, and associated benefits expressed in quality-adjusted life-years of the patient population were calculated. When patients responded according to the predicted clinical pathway presented in health technology assessment reports (scenario B), implementing outcome-based reimbursement models (models 2 and 3) had lower associated budget impacts while gaining similar benefits compared with the discount (scenario 1, €8.9 million to €6.6 million vs €9.2 million). In the case of unstable responders (scenario C), costs for payers are lower in the outcome-based scenarios (€4.1 million and €3.0 million, scenario 2C and 3C, respectively) compared with implementing the discount (€9.2 million, scenario 1C). Outcome-based models can mitigate the financial risk of reimbursing atidarsagene autotemcel. This can be considerably beneficial over simple discounts when clinical performance was similar to or worse than predicted. • High-priced one-off gene therapies, such as atidarsagene autotemcel (AA, Libmeldy), are innovative treatments that often fulfill an unmet medical need. However, they represent a reimbursement challenge to many healthcare systems because of substantial upfront costs, which are often incurred all at once, with the claim of durability of effects. • More guidance is needed to inform decision makers and healthcare payers which type of reimbursement and payment model is suitable. Therefore, a framework and calculation tool were used to help decision makers weigh the different payment scenarios for AA and help them negotiate and implement an outcome-based spread payment model. • The results from this calculation tool demonstrated that for AA, outcome-based models could considerably mitigate the financial risks for the payer in cases when clinical performance was similar or worse than predicted. The exact outcomes of the calculations should be treated with caution as the discount rates used are arbitrary and should be updated with values from decision makers. [ABSTRACT FROM AUTHOR]

Cell-based medicinal products (CBMPs) are a growing class of therapeutics that promise new treatments for complex and rare diseases. Given the inherent complexity of the whole human cells comprising CBMPs, there is a need for robust and fast analytical methods for characterization, process monitoring, and quality control (QC) testing during their manufacture. Existing techniques to evaluate and monitor cell quality typically constitute labor-intensive, expensive, and highly specific staining assays. In this work, we combine image-based deep learning with flow imaging microscopy (FIM) to predict cell health metrics using cellular morphology "fingerprints" extracted from images of unstained Jurkat cells (immortalized human T-lymphocyte cells). A supervised (i.e., algorithm trained with human-generated labels for images) fingerprinting algorithm, trained on images of unstained healthy and dead cells, provides a robust stain-free, non-invasive, and non-destructive method for determining cell viability. Results from the stain-free method are in good agreement with traditional stain-based cytometric viability measurements. Additionally, when trained with images of healthy cells, dead cells and cells undergoing chemically induced apoptosis, the supervised fingerprinting algorithm is able to distinguish between the three cell states, and the results are independent of specific treatments or signaling pathways. We then show that an unsupervised variational autoencoder (VAE) algorithm trained on the same images, but without human-generated labels, is able to distinguish between samples of healthy, dead and apoptotic cells along with cellular debris based on learned morphological features and without human input. With this, we demonstrate that VAEs are a powerful exploratory technique that can be used as a process monitoring analytical tool. [ABSTRACT FROM AUTHOR]

Mesenchymal Stem Cells (MSCs) are of interest in the clinic because of their immunomodulation capabilities, capacity to act upstream of inflammation, and ability to sense metabolic environments. In standard physiologic conditions, they play a role in maintaining the homeostasis of tissues and organs; however, there is evidence that they can contribute to some autoimmune diseases. Gaining a deeper understanding of the factors that transition MSCs from their physiological function to a pathological role in their native environment, and elucidating mechanisms that reduce their therapeutic relevance in regenerative medicine, is essential. We conducted a Systematic Review and Meta-Analysis of human MSCs in preclinical studies of autoimmune disease, evaluating 60 studies that included 845 patient samples and 571 control samples. MSCs from any tissue source were included, and the study was limited to four autoimmune diseases: multiple sclerosis, rheumatoid arthritis, systemic sclerosis, and lupus. We developed a novel Risk of Bias tool to determine study quality for in vitro studies. Using the International Society for Cell & Gene Therapy's criteria to define an MSC, most studies reported no difference in morphology, adhesion, cell surface markers, or differentiation into bone, fat, or cartilage when comparing control and autoimmune MSCs. However, there were reported differences in proliferation. Additionally, 308 biomolecules were differentially expressed, and the abilities to migrate, invade, and form capillaries were decreased. The findings from this study could help to explain the pathogenic mechanisms of autoimmune disease and potentially lead to improved MSC-based therapeutic applications. • MSCs from autoimmune patients have increased levels of IL-6. • MSCs from autoimmune patients have a reduced ability to migrate and invade. • MSCs from autoimmune patients have a reduced ability to form capillaries. • MSCs from autoimmune disease are still adherent, spindle-shaped, and capable of differentiation. [ABSTRACT FROM AUTHOR]

Studies of the dark genome are providing important insights into human pain insensitivity and chronic pain conditions. Long noncoding RNAs (lncRNAs), a major portion of the dark genome, have diverse cellular functions and can dynamically regulate pain thresholds. Often dismissed as merely fine tuners of gene expression, some lncRNAs instead play a major role in chronic pain mechanisms. Precise spatial and temporal expression patterns and low expression levels make some lncRNAs viable analgesic drug targets. RNA-targeted precision therapeutics may have fewer side effects than targeting broadly and highly expressed proteins. The dark genome, the nonprotein-coding part of the genome, is replete with long noncoding RNAs (lncRNAs). These functionally versatile transcripts, with specific temporal and spatial expression patterns, are critical gene regulators that play essential roles in health and disease. In recent years, FAAH-OUT was identified as the first lncRNA associated with an inherited human pain insensitivity disorder. Several other lncRNAs have also been studied for their contribution to chronic pain and genome-wide association studies are frequently identifying single nucleotide polymorphisms that map to lncRNAs. For a long time overlooked, lncRNAs are coming out of the dark and into the light as major players in human pain pathways and as potential targets for new RNA-based analgesic medicines. [ABSTRACT FROM AUTHOR]

Preclinical and clinical studies indicate that immune responses against HSV-1 vectors, oncolytic or non-replicative, do not constitute a major impediment to their efficacy or safety. Despite oHSV replication in tumors and their elicited immune responses, they have proved to be very safe and well-tolerated in cancer patients. In contrast to early safety concerns, current non-replicative gene therapy vectors, which often express only the therapeutic transgene(s), are safe and elicit only limited immune responses. Seropositivity and immune responses against HSV do not lead to the elimination of either HSV or infected cells, and the vectors can be re-administered several times. Three HSV-1 vectors have already been approved, two for cancer therapy and one for gene therapy. Other HSV-1 vectors are currently being clinically tested, and serious adverse events linked to the vector have not been observed. Herpes simplex virus type 1 (HSV-1) is a DNA virus and human pathogen used to construct promising therapeutic vectors. HSV-1 vectors fall into two classes: replication-selective oncolytic vectors for cancer therapy and defective non-replicative vectors for gene therapy. Vectors from each class can accommodate ≥30 kb of inserts, have been approved clinically, and demonstrate a relatively benign safety profile. Despite oncolytic HSV (oHSV) replication in tumors and elicited immune responses, the virus is well tolerated in cancer patients. Current non-replicative vectors elicit only limited immune responses. Seropositivity and immune responses against HSV-1 do not eliminate either the vector or infected cells, and the vectors can therefore be re-administered. In this review we highlight vectors that have been translated to the clinic and host–virus immune interactions that impact on the safety and efficacy of HSVs. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) have become the delivery medium of choice for a variety of genomic medicine applications i.e., gene therapy, gene editing/regulation, and ex-vivo cell therapy. AAVs are protein-DNA complexes which have unique stability characteristics that are susceptible to various stress exposure conditions commonly seen in the drug product (DP) life cycle. This review takes a comprehensive look at AAV DP formulation and process development considerations that could impact critical quality attributes (CQAs) during manufacturing, packaging, shipping, and clinical use. Additional aspects related to AAV development reviewed herein are: (1) Different AAV serotypes with unique protein sequences and charge characteristics potentially leading to discrete stability profiles; (2) Manufacturing process challenges and optimization efforts to improve yield, recovery and purity especially during early development activities; and (3) Defining and identifying CQAs with analytical methods which are constantly evolving and present unique characterization challenges for AAV-based products. [Display omitted] [ABSTRACT FROM AUTHOR]

• Two image analysis methods have been developed for viral vector characterization, and their performances were compared. • A combination of cryo-electron microscopy and image analysis enables the accurate assessment of the viral vector population. • Convolutional neural networks and morphological analysis quantified exiguous impurities in viral vector products with 90% purity. • The utility of convolutional neural networks to characterize wider adeno-associated viral products was demonstrated. • Image analysis methods based on cryo-electron micrographs might evaluate the slight difference in the viral vector population. Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products. [ABSTRACT FROM AUTHOR]

Metachromatic leukodystrophy (MLD) is a fatal, progressive neurodegenerative disorder caused by biallelic pathogenic mutations in the ARSA (Arylsulfatase A) gene. With the advent of presymptomatic diagnosis and the availability of therapies with a narrow window for intervention, it is critical to define a standardized approach to diagnosis, presymptomatic monitoring, and clinical care. To meet the needs of the MLD community, a panel of MLD experts was established to develop disease-specific guidelines based on healthcare resources in the United States. This group developed a consensus opinion for best-practice recommendations, as follows: (i) Diagnosis should include both genetic and biochemical testing; (ii) Early diagnosis and treatment for MLD is associated with improved clinical outcomes; (iii) The panel supported the development of newborn screening to accelerate the time to diagnosis and treatment; (iv) Clinical management of MLD should include specialists familiar with the disease who are able to follow patients longitudinally; (v) In early onset MLD, including late infantile and early juvenile subtypes, ex vivo gene therapy should be considered for presymptomatic patients where available; (vi) In late-onset MLD, including late juvenile and adult subtypes, hematopoietic cell transplant (HCT) should be considered for patients with no or minimal disease involvement. This document summarizes current guidance on the presymptomatic monitoring of children affected by MLD as well as the clinical management of symptomatic patients. Future data-driven evidence and evolution of these recommendations will be important to stratify clinical treatment options and improve clinical care. [ABSTRACT FROM AUTHOR]

Gene therapy is a fast-growing field showing great potential to treat genetic diseases and cancer. With accelerating gene therapy development and approval, their environment risk assessment (ERA) becomes increasingly important. An ERA is an assessment of the risks to human health and the environment upon exposure to a medicinal product as the result of its release during clinical development or after entering the market. Because ERA is an important component of regulatory submission, drug developers must perform a robust assessment to ensure the safety of unintended persons, animal, plants, microorganisms and environment at large. Global regulations on gene therapy ERA continue to evolve. Gene therapy ERAs are carried out according to general principles as provided in regulatory guidelines for application of clinical trials and marketing authorizations. The current review intends to summarize regulations and content requirements on gene therapy ERA in European Union, the USA and Japan. The approved gene therapy products by EMA and US Food and Drug Administration are analyzed for the critical aspects of their ERAs to provide the current status and practice of gene therapy ERAs by drug developers. For this purpose, the main contents of these gene therapy ERAs are summarized. Critical safety factors of gene therapy ERAs are described. With more experience and knowledge to be accumulated, gene therapy ERAs are expected to be less challenging with commonly used viral vectors. [ABSTRACT FROM AUTHOR]

With the increase in cell and gene therapy (CGT) clinical trials in recent years has come a subsequent increase in the number of contract development and manufacturing organizations (CDMOs). Successful transition from development and early-phase clinical trials to commercialization of a CGT product often depends on selecting the best-suited CDMO. However, many CGT companies are small biotech companies that lack expertise in the field or do not have experience selecting and transferring a process to a CDMO. Given the interest in this topic, a roundtable with CGT developers and CDMO members at the 2023 annual meeting of the International Society of Cell and Gene Therapy Paris discussed these critical aspects of product development, including technical expertise, risk sharing and timing of partnerships. Here, we'll analyze the considerations discussed by the panel and elaborate on other factors crucial for CGT development. [ABSTRACT FROM AUTHOR]

To better understand the attitudes and behaviors of investors involved in funding cell and gene therapy (CGT) businesses, the Business Development and Finance) subcommittee of International Society for Cell and Gene Therapy, in collaboration with Truist Securities, conducted a broad survey of the investment community in late 2021. This survey follows a similar study that this group executed in 2018, and the longitudinal comparisons between the two time periods provide insights into how investor behavior in the CGT field has evolved. The vast majority of investor respondents are specialist biotech investors who are primarily active in deploying capital in North America and Europe. There was a notable increase in the proportion of investors actively deploying capital in China and Japan between 2018 and 2021. The percentage of respondents' portfolios dedicated to CGT companies has also increased in this period, reflecting a noteworthy trend in the therapeutic landscape. Clinically significant data remain the dominant force behind investment decisions, whereas competition from other drug modalities has now emerged as the most-cited barrier to making a CGT investment, eclipsing safety concerns as the most significant barrier to investment in 2018. Concerns around manufacturing and scale-up have also increased in prominence amongst the investment community. Gene-editing technologies are attracting investors as the most compelling new CGT technology. This survey also revealed that most investors expect to increase their level of investment in allogeneic technologies relative to autologous products in the coming years. [ABSTRACT FROM AUTHOR]

Cell and gene therapy poses evolving challenges. The current article summarizes the discussions held by European Regional Committee of the International Society for Cell & Gene Therapy and the European Society for Blood and Marrow Transplantation (EBMT) on the current challenges in this field, focusing on the European setting. This article emphasizes the imperative assessment of real-world cell and gene therapy activity, advocating for expanded registries beyond hematopoietic transplantation and chimeric antigen receptor–T-cell therapy. Accreditation's role in ensuring standardized procedures, as exemplified by JACIE (The Joint Accreditation Committee of ISCT-Europe and EBMT), is crucial for safety. Access to commercial products and reimbursement variations among countries underscore the need for uniform access to advanced therapy medical products (ATMPs). Academic product development and point-of-care manufacturing face barriers to patient access. Hospital Exemption's potential, demonstrated by some initial experiences, may increase patient accessibility in individual situations. Regulatory challenges, including the ongoing European ATMPs legislation review, necessitate standardized criteria for Hospital Exemption and mandatory reporting within registries. Efforts to combat unproven therapies and fraud involve collaboration between scientific societies, regulatory bodies and patient groups. Finally, is important to highlight the vital role of education and workforce development in meeting the escalating demand for specialized professionals in the ATMP field. Collaboration among scientific societies, academic institutions, industry, regulatory bodies and patient groups is crucial for overcoming all these challenges to increase gene and cell therapy activity in Europe. [ABSTRACT FROM AUTHOR]

There is lack of guidance for immune monitoring and infection prevention after administration of ex vivo genetically modified hematopoietic stem cell therapies (GMHSCT). We reviewed current infection prevention practices as reported by providers experienced with GMHSCTs across North America and Europe, and assessed potential immunologic compromise associated with the therapeutic process of GMHSCTs described to date. Based on these assessments, and with consensus from members of the International Society for Cell & Gene Therapy (ISCT) Stem Cell Engineering Committee, we propose risk-adapted recommendations for immune monitoring, infection surveillance and prophylaxis, and revaccination after receipt of GMHSCTs. Disease-specific and GMHSCT-specific considerations should guide decision making for each therapy. [ABSTRACT FROM AUTHOR]

Gene replacement therapy (onasemnogene abeparvovec) is associated with an improvement of the prognosis of children with spinal muscular atrophy, but information on long-term respiratory outcome is scarce. The aim of this study was to report the polysomnography findings and respiratory muscle function of infants with treatment-naive spinal muscular atrophy type 1 and 2 up to 24 months after onasemnogene abeparvovec monotherapy. A clinical and motor evaluation, respiratory muscle function testing, and polysomnography were performed repeatedly. Fifteen spinal muscular atrophy patients (1 presymptomatic, 7 type 1b, 6 type 1c, and 1 type 2) were included at a median age of 8.6 months (range 3.8–12.6) and followed for 24 months. The thoracic over head circumference ratio was close to normal at baseline (median 1.00 (range 0.90–1.05)) and increased significantly over time. All polysomnography and nocturnal gas exchange parameters were within normal ranges at baseline (median apnea-hypopnea index 2.5 events/hour (range 0.4–5.3)) and follow-up. The inspiratory muscle strength was normal at baseline but tended to slightly decrease over time and the expiratory muscle strength was low at any time especially for patients with recurrent respiratory infections (median (range) at baseline in cmH 2 O: crying esophageal pressure 54 (30–110), crying transdiaphragmatic pressure 65 (35–107), gastric pressure during maximal cough 26 (10–130), esophageal pressure during maximal cough 61 (38–150)). Only 3 patients required noninvasive ventilation. A continuous respiratory monitoring of spinal muscular atrophy patients during the first years of life following onasemnogene abeparvovec monotherapy seems recommended despite the normality of polysomnography parameters. • Fifteen patients received gene replacement therapy as first and only treatment. • Thoracic over head circumference was close to normal and increased over time. • All polysomnography parameters were within normal ranges at all time. • The inspiratory muscle strength was normal but the expiratory muscles were weak. • We recommend a continuous respiratory monitoring after gene therapy. [ABSTRACT FROM AUTHOR]

Gene delivery has revolutionized conventional medical approaches to vaccination, cancer, and autoimmune diseases. However, current gene delivery methods are limited to either intravenous administration or direct local injections, failing to achieve well biosafety, tissue targeting, drug retention, and transfection efficiency for desired therapeutic outcomes. Transdermal drug delivery based on various delivery strategies can offer improved therapeutic potential and superior patient experiences. Recently, there has been increased foundational and clinical research focusing on the role of the transdermal route in gene delivery and exploring its impact on the efficiency of gene delivery. This review introduces the recent advances in transdermal gene delivery approaches facilitated by drug formulations and medical devices, as well as discusses their prospects. [Display omitted] [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is one of the most common genetic diseases and was, until recently, a leading genetic cause of infant mortality. Three disease-modifying treatments have dramatically changed the disease trajectories and outcome for severely affected infants (SMA type 1), especially when initiated in the presymptomatic phase. One of these treatments is the adeno-associated viral vector 9 (AAV9) based gene therapy onasemnogene abeparvovec (Zolgensma®), which is delivered systemically and has been approved by the European Medicine Agency for SMA patients with up to three copies of the SMN2 gene or with the clinical presentation of SMA type 1. While this broad indication provides flexibility in patient selection, it also raises concerns about the risk-benefit ratio for patients with limited or no evidence supporting treatment. In 2020, we convened a European neuromuscular expert working group to support the rational use of onasemnogene abeparvovec, employing a modified Delphi methodology. After three years, we have assembled a similar yet larger group of European experts who assessed the emerging evidence of onasemnogene abeparvovec's role in treating older and heavier SMA patients, integrating insights from recent clinical trials and real-world evidence. This effort resulted in 12 consensus statements, with strong consensus achieved on 9 and consensus on the remaining 3, reflecting the evolving role of onasemnogene abeparvovec in treating SMA. • Updated European consensus on the use of onasemnogene abeparvovec for SMA treatment. • Emphasis on the importance of early intervention for improved patient outcomes. • Recommendations include considerations for older and heavier patients. • Data collection for safety and effectiveness is critical for treatment optimization. • New statement advocating for newborn screening implementation for SMA. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a neuromuscular disease, causing progressive muscle weakness due to loss of lower motoneurons. Since 2017, three therapies, two modifying gene transcription and one adding the defective gene, have been approved with comparable efficacy on motor outcome. Data on cognitive outcomes of treated SMA type 1 patients is limited. The aim of this study was to evaluate cognitive function in symptomatic and presymptomatic SMA type 1 patients with two or three SMN2 copies who received SMN-modifying or gene-addition therapy in the first year of life. Cognitive testing was performed in 20 patients, including 19 symptomatic SMA type 1 patients with up to three SMN2 copies and 1 pre-symptomatically treated patient. Children were tested using Bayley Scales of Infant Development (BSID-III) at the age of 2 or 3 years or the Wechsler Preschool and Primary Scale of Intelligence (WPSII-IV) at the of age of 5 years. 11/20 patients showed subnormal cognitive development. Boys had significantly lower cognitive scores. Patients requiring assisted ventilation or feeding support were more likely to have cognitive deficits. Achieving more motor milestones was associated with a better cognitive outcome. Treated patients with SMA type 1 have heterogeneous cognitive function with 55 % of patients showing deficits. Risk factors for cognitive impairment in our cohort were male gender and need for assisted ventilation or feeding support. Therefore, cognitive assessment should be included in the standard of care to allow early identification of deficits and potential therapeutic interventions. • Data on cognitive function in SMA type 1 patients is limited. • 55 % of tested SMA patients showed cognitive impairment. • Boys scored significantly lower than girls. • Cognitive testing should be implemented in standard care of SMA patients. [ABSTRACT FROM AUTHOR]

Combination immunotherapy has shown promising potential for enhancing the objective response rate compared to immune checkpoint blockade (ICB) monotherapy. However, combination therapy with multi-drugs is limited by the different properties of the agents and inconsistent synergistic targeted delivery. Herein, based on a universal triterpene template and the anticancer active agent ursolic acid (UA), a cytomembrane-coated biomimetic delivery nanoplatform (UR@M) prepared by the self-assembly of a PD-L1 targeted CRISPR/Cas9 system and UA was designed for hepatocellular carcinoma (HCC) treatment. UR@M showed enhanced tumor accumulation in vivo with homologous tumor targeting, and CRISPR in the nanosystem exhibited potent gene-editing efficiency of 76.53% in vitro and 62.42% in vivo with no off-target effects. UA activated the natural immune system through the TLR-2-MyD88-TRAF6 pathway, which synergistically enhanced the proliferation of natural killer cells and dendritic cells and realized excellent immune cytotoxic T cell infiltration by combining with the ICB of PD-L1. The strategy of work along both lines based on innate immune and adaptive immunity displayed a significant effect in tumor regression. Overall, the UA-templated strategy "killed three birds with one stone" by establishing a self-assembly nanosystem, inducing tumor cell death, and promoting synergistic immunostimulation for HCC treatment. A biomimetic nanodrug UR@M from the triterpenoids-templated self-assembly is developed to deliver CRISPR/Cas9, exhibiting synergistic immunotherapy by activating innate immune by TLR-2 pathway and gene therapy of PD-L1 by CRISPR/Cas9. [Display omitted] [ABSTRACT FROM AUTHOR]

Inherited genetic disorders of the liver pose a significant public health burden. Liver transplantation is often limited by the availability of donor livers and the exorbitant costs of immunosuppressive therapy. To overcome these limitations, nucleic acid therapy provides a hopeful alternative that enables gene repair, gene supplementation, and gene silencing with suitable vectors. Though viral vectors are the most efficient and preferred for gene therapy, pre-existing immunity debilitating immune responses limit their use. As a potential alternative, lipid nanoparticle-mediated vectors are being explored to deliver multiple nucleic acid forms, including pDNA, mRNA, siRNA, and proteins. Herein, we discuss the broader applications of lipid nanoparticles, from protein replacement therapy to restoring the disease mechanism through nucleic acid delivery and gene editing, as well as multiple preclinical and clinical studies as a potential alternative to liver transplantation. Lipid nanoparticles serve as delivery vehicles to transport therapeutic genes or gene-editing tools into target cells, which allows for correction of genetic mutations, introduction of therapeutic genes, or modulation of gene expression. [Display omitted] [ABSTRACT FROM AUTHOR]

Optimum genetic delivery for modulating target genes to diseased tissue is a major obstacle for profitable gene therapy. Lipid nanoparticles (LNPs), considered a prospective vehicle for nucleic acid delivery, have demonstrated efficacy in human use during the COVID-19 pandemic. This study introduces a novel biomaterial-based platform, M1-polarized macrophage-derived cellular nanovesicle-coated LNPs (M1-C-LNPs), specifically engineered for a combined gene-immunotherapy approach against solid tumor. The dual-function system of M1-C-LNPs encapsulates Bcl2 -targeting siRNA within LNPs and immune-modulating cytokines within M1 macrophage-derived cellular nanovesicles (M1-NVs), effectively facilitating apoptosis in cancer cells without impacting T and NK cells, which activate the intratumoral immune response to promote granule-mediating killing for solid tumor eradication. Enhanced retention within tumor was observed upon intratumoral administration of M1-C-LNPs, owing to the presence of adhesion molecules on M1-NVs, thereby contributing to superior tumor growth inhibition. These findings represent a promising strategy for the development of targeted and effective nanoparticle-based cancer genetic-immunotherapy, with significant implications for advancing biomaterial use in cancer therapeutics. M1-polarized macrophage-derived cellular nanovesicle-coated lipid nanoparticles (M1-C-LNPs) were designed for combined gene and immunotherapy against solid tumors, with superior tumor inhibition efficacy and safety from targeted delivery and immune activation. [Display omitted] [ABSTRACT FROM AUTHOR]

It is crucial to develop a long-term therapy that targets hemophilia A and B, including inhibitor-positive patients. We have developed an Adeno-associated virus (AAV) based strategy to integrate the bypass coagulation factor, activated FVII (murine, mFVIIa) gene into the Rosa26 locus using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 mediated gene-editing. AAV vectors designed for expression of guide RNA (AAV8-gRNA), Cas9 (AAV2 neddylation mutant-Cas9), and mFVIIa (AAV8-mFVIIa) flanked by homology arms of the target locus were validated in vitro. Hemophilia B mice were administered with AAV carrying gRNA, Cas9 (1 × 1011 vgs/mouse), and mFVIIa with homology arms (2 × 1011 vgs/mouse) with appropriate controls. Functional rescue was documented with suitable coagulation assays at various time points. The data from the T7 endonuclease assay revealed a cleavage efficiency of 20–42 %. Further, DNA sequencing confirmed the targeted integration of mFVIIa into the safe-harbor Rosa26 locus. The prothrombin time (PT) assay revealed a significant reduction in PT in mice that received the gene-editing vectors (22 %), and a 13 % decline in mice that received only the AAV-FVIIa when compared to mock treated mice, 8 weeks after vector administration. Furthermore, FVIIa activity in mice that received triple gene-editing vectors was higher (122.5mIU/mL vs 28.8mIU/mL) than the mock group up to 15 weeks post vector administration. A hemostatic challenge by tail clip assay revealed that hemophilia B mice injected with only FVIIa or the gene-editing vectors had significant reduction in blood loss. In conclusion, AAV based gene-editing facilitates sustained expression of coagulation FVIIa and phenotypic rescue in hemophilia B mice. AAV mediated gene editing using CRISPR/Cas9 facilitates integration of a bypass coagulation factor VIIa and rescues bleeding phenotype in hemophilia B mice. Image created using Biorender.com. [Display omitted] • CRISPR/Cas9 mediated targeting of Rosa26 locus was found to have 20-42% efficiency in vitro. • Successful insertion of murine activated factor VII (mFVIIa) into the Rosa26 locus was achieved by CRISPR/Cas9 gene editing. • AAV mediated knock-in of bypass clotting factor mFVIIa using CRISPR/Cas9 mitigated bleeding phenotype in hemophilia B mice. • Increased FVIIa activity and shortened prothrombin time was attained after gene editing therapy in hemophilia B mice. [ABSTRACT FROM AUTHOR]

• DMD is caused by DMD gene mutations that result in a lack of dystrophin protein. • BMD is also caused by DMD mutations, but symptoms are milder compared with DMD. • We present a case study of a patient with BMD and his affected relatives. • The patient could walk at age 61 years, despite 46% of his DMD gene being missing. • These findings informed minigene constructs, a promising therapy option for DMD. Duchenne muscular dystrophy is a neuromuscular disease caused by DMD gene mutations that result in an absence of functional dystrophin protein. Patients with Duchenne experience progressive muscle weakness, are typically wheelchair dependent by their early teens, and develop respiratory and cardiac complications that lead to death in their twenties or thirties. Becker muscular dystrophy is also caused by DMD gene mutations, but symptoms are less severe and progression is slower compared with Duchenne. We describe a case study of a patient with Becker muscular dystrophy who was still ambulant at age 61 years and had a milder phenotype than Duchenne, despite 46% of his DMD gene being missing. His affected relatives had similarly mild phenotypes and clinical courses. These data guided the understanding of the criticality of various regions of dystrophin and informed the development of micro-dystrophin constructs to compensate for the absence of functional dystrophin in Duchenne. [ABSTRACT FROM AUTHOR]

The tumor microenvironment is importantly shaped by various cytokines, where interleukins (ILs) and interferons (IFNs) shape the balance of immune activity within tumor niche and associated lymphoid organs. Their importance in activation and tuning of both innate and adaptive immune responses prompted their use in several clinical trials, albeit with limited therapeutic efficacy and risk of toxicity due to systemic administration. Increasing preclinical evidence suggests that local delivery of ILs and IFNs could significantly increase their effectiveness, while simultaneously attenuate the known side effects and issues related to their biological activity. A prominent way to achieve this is to use cell-based delivery vehicles. For this purpose, mesenchymal stromal stem cells (MSCs) are considered an almost ideal candidate. Namely, MSCs can be obtained in large quantities and from obtainable sources (e.g. umbilical cord or adipose tissue), their ex vivo expansion is relatively straightforward compared to other cell types and they possess very low immunogenicity making them suitable for allogeneic use. Importantly, MSCs have shown an intrinsic capacity to respond to tumor-directed chemotaxis. This review provides a focused and detailed discussion on MSC-based gene therapy using ILs and IFNs, engineering techniques and insights on potential future advancements. [Display omitted] • MSCs represent one of the most ideal candidates for intratumoral cytokine delivery. • Both ILs and IFNs importantly shape the tumor microenvironment. • Gene delivery of ILs and IFNs can favourably shape the tumor immune contexture. • MSCs can be modified to gain synergistic anti-tumor effects and to inhibit tumor promoting activities of certain cytokines. [ABSTRACT FROM AUTHOR]

Workforce education and development are key cornerstones in advancing and maturing the Cell & Gene Therapy sector. A skilled worker shortage can significantly impact and delay progress as well as the quality of output for any developer, thereby negatively impacting a patient's access to life-saving treatments. Several roundtable discussions were held at the International Society for Cell & Gene Therapy (ISCT) 2023 Annual Meeting to dive deeper into the current state of workforce development and solutions to address this bottleneck. One roundtable discussion was co-hosted by the Alliance for Regenerative Medicine (ARM) and ISCT, which focused on the gap analysis provided for the United States Cell & Gene Therapy (CGT) sector, highlighting the lack of skilled workers in manufacturing and quality control. In this manuscript, the roundtable participants continue this conversation, review the roles and staffing requirements in both academic and industry as well as small and large company settings. The adoption of increased manufacturing automation is one promising solution to propel the sector forward. However, automation alone won't replace on-site staff, but will lower the bar to entry for a larger pool of people and require different training. This paper also addresses the workforce development and training paradigm shift as advanced manufacturing techniques are implemented, which will differ considerably based on the type of manufacturing efforts, thus emphasizing the need for a well-thought-out strategy to up-skill and re-skill the technical workforce to adapt to these advancements. Organizations such as ISCT and ARM have a role to play in propelling the field forward, providing awareness and education to stakeholders at all levels, as well as acting as a convener and participating as a key stakeholder in discussions and partnerships between academia and industry towards solutions for training the best personnel for CGT manufacturing. This scope includes novel digital tools and technologies to simplify training to increase access to new talent pools interested in careers in a rapidly advancing sector. [ABSTRACT FROM AUTHOR]

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting. [ABSTRACT FROM AUTHOR]

Allogeneic hemopoietic cell transplantation remains the goal of therapy for high-risk acute myeloid leukemia (AML). However, treatment failure in the form of leukemia relapse or severe graft-versus-host disease remains a critical area of unmet need. Recently, significant progress has been made in the cell therapy-based interventions both before and after transplant. In this review, the Stem Cell Engineering Committee of the International Society for Cell and Gene Therapy summarizes the literature regarding the identification of high risk in AML, treatment approaches before transplant, optimal transplant platforms and measures that may be taken after transplant to ideally prevent, or, if need be, treat AML relapse. Although some strategies remain in the early phases of clinical investigation, they are built on progress in pre-clinical research and cellular engineering techniques that are already improving outcomes for children and adults with high-risk malignancies. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a progressive autoimmune disease and drug therapy has been restricted due to poor therapeutic efficacy and adverse effects. In RA synovium, dendritic cells present self-antigens to activate cascade immune pathway. Furthermore, downstream macrophages secrete high levels of pro-inflammatory cytokines; Hyperplasia of activated synovial fibroblasts (FLS) is responsible for hypoxic synovium microenvironment, secretion of cytokines/chemokines and erosion of bone/cartilage tissues. Positive feedback loop of inflammation between macrophages and FLS independent of antigen-presentation is constructed. Herein, an injectable pH-sensitive peptide hydrogel encapsulating siRNA/Methotrexate-polyethyleneimine (siMP, including sip65MP, sip38MP, siCD86MP) and Bismuthene nanosheet/Methotrexate-polyethyleneimine (BiMP) is successfully developed. Among them, siCD86MP reduces protein level of co-stimulatory molecule CD86 while sip65MP and sip38MP separately inhibit NF-κB and MAPK-p38 pathways of macrophages and FLS to suppress secretion of cytokines and MMPs. Meanwhile, reduction in anti-apoptotic property of FLS induced by inhibition of NF-κB pathway has a synergistic effect with photodynamic therapy (PDT) and photothermal therapy (PTT) mediated by BiMP for FLS elimination, effectively ameliorating hypoxic synovium microenvironment. After being injected into synovium, hydrogel responds to acidic microenvironment and serves as a reservoir for sustained drug release and inherent retention capacity of which enables cationic nanoparticles to bypass tissue barrier for precise synovium targeting. This brand-new drug delivery system combines modulating cascade immune pathway from beginning to end by RNAi and eliminating FLS for improving synovium microenvironment by phototherapy together, providing a robust strategy for clinical RA treatment. In RA synovium, dendritic cells present self-antigens to activate the whole inflammatory pathway. Inhibiting antigen-presentation could effectively block the initiation of entire pathway. However, the downstream macrophages secrete high levels of pro-inflammatory cytokines, while excessively proliferated synovial fibroblasts (FLS) are responsible for hypoxia microenvironment, cytokine/chemokine release, bone/cartilage erosion, thus constructing an immune cycle independent of antigen-presentation together with macrophages. In this timely research, we successfully developed an implantable and pH-sensitive peptide hydrogel loading siRNA/methotrexate-polyethyleneimine (siMP: siCD86MP, sip65MP, sip38MP) and bismuthene nanosheet/methotrexate-polyethyleneimine (BiMP). siCD86MP can reduce the protein level of co-stimulatory molecule CD86 to block the initiation of inflammatory pathway. sip65MP and sip38MP can inhibit NF-κB and MAPK-p38 pathways in macrophages and FLS to ameliorate secretion of cytokines and MMPs, fundamentally blocking inflammatory circulation. Meanwhile, reduction in anti-apoptosis property of FLS induced by siMP had the synergistic effect with PDT and PTT of BiMP for FLS elimination, effectively ameliorating synovium microenvironment. After injection into synovium, the hydrogel responded to the acidic microenvironment and served as a drug reservoir for sustained drug release, while reducing systematic toxicity of cationic nanoparticles. The inherent retention capacity of hydrogel enabled nanoparticles to bypass the tissue barrier and achieve precise targeting. This brand-new drug delivery system combines modulating inflammation pathways from beginning to end by RNAi and eliminating FLS by phototherapy for improving synovium microenvironment together, providing a robust strategy for RA treatment. [Display omitted] • siRNA modulated cascade immune activation through inhibiting upstream antigen-presentation and downstream inflammatory circulation. • Bismuthene nanosheets exerted efficient PDT&PTT therapy to eliminate FLS for alleviating hypoxic synovium microenvironment. • Modulation of cascade immune activation and alleviating hypoxia microenvironment can synergistically treat RA. • Nap Hydrogel possessed excellent biocompatibility, injectability, self-healing, pH responsiveness, synovium targeting and retention. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Although clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing holds immense potential for genetic manipulation, its clinical application is hindered by the absence of an efficient heart-targeted drug delivery system. Herein, we developed CRISPR-Cas9 ribonucleoprotein (RNP)-loaded extracellular vesicles (EVs) conjugated with cardiac-targeting peptide (T) for precise cardiac-specific genome editing. RNP complexes containing Cas9 and single guide RNA targeting miR-34a, an MI-associated molecular target, were loaded into EVs (EV@RNP). Gene editing by EV@RNP attenuated hydrogen peroxide-induced apoptosis in cardiomyocytes via miR-34a inhibition, evidenced by increased B-cell lymphoma 2 levels, decreased Bcl-2-associated X protein levels, and the cleavage of caspase-3. Additionally, to improve cardiac targeting in vivo , we used click chemistry to form functional T-EV@RNP by conjugating T peptides to EV@RNP. Consequently, T-EV@RNP-mediated miR-34a genome editing might exert a protective effect against MI, reducing apoptosis, ameliorating MI injury, and facilitating the recovery of cardiac function. In conclusion, the genome editing delivery system established by loading CRISPR/Cas9 RNP with cardiac-targeting EVs is a powerful approach for precise and tissue-specific gene therapy for cardiovascular disease. [Display omitted] [ABSTRACT FROM AUTHOR]

Delivering functional gene into targeted skin cells or tissues to modulate the genes expression, has the potential to treat various hereditary cutaneous disorders. Nevertheless, the lack of safe and effective gene delivery vehicles greatly limits the clinical translation of gene therapy for inherited skin diseases. Herein, we developed a facile elution fractionation strategy to isolate eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0 from the one crude HPAE 23.7k , and investigated the expression efficiency for TGM1 and COL7A1 plasmids. Gene transfection results revealed that the intermediate MW HPAEs, HPAE 20.6k , exhibited the highest gene transfection efficiency (46.4%) and the strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product. Importantly, best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAEs gene delivery vectors, expediting their clinical translation in inherited skin diseases. HPAEs crude polymer is fractionated to eight HPAEs with M w ranging from 7.6 to 131.8 kg/mol and Đ < 2.0. The best-performing isolated HPAE effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of type VII collagen (C7) and transglutaminase-1 proteins in cutaneous cells. [Display omitted] • We establish a straightforward step-by-step elution fractionation strategy to isolate HPAEs with different molecular weight. • Isolated intermediate molecular weight HPAEs exhibit high transfection efficiency and biocompatibility in diverse cell types. • The top-performing HPAEs efficiently deliver TGM and COL7A1 DNA to potentially rescue hereditary cutaneous disorders. [ABSTRACT FROM AUTHOR]

Hemophilia A and B are rare X-linked genetic bleeding disorders due to a complete or partial deficiency in the coagulation factors VIII or IX, respectively. The main treatment for hemophilia is prophylactic and based on coagulation factor replacement therapies. These treatments have significantly reduced bleeding and improved the patients' quality of life. Nevertheless, repeated joint bleedings (hemarthroses), even subclinical hemarthroses, can lead to hemophilic arthropathy (HA). This disabling condition is characterized by chronic pain due to synovial inflammation, cartilage and bone destruction requiring ultimately joint replacement. HA resembles to rheumatoid arthritis because of synovitis but HA is considered as having similarities with osteoarthritis as illustrated by the migration of immune cells, production of inflammatory cytokines, synovial hypertrophy and cartilage damage. Various drugs have been evaluated for the management of HA with limited success. The objective of the review is to discuss new therapeutic approaches with a special focus on the studies that have investigated the potential of using mesenchymal stromal cells (MSCs) in the management of HA. A systematic review of the literature has been made. Most of the studies have focused on the interest of MSCs for the delivery of missing factors VIII or IX but in some studies, more insight on the effect of MSC injection on synovial inflammation or cartilage structure were provided and put in perspective for possible clinical applications. [ABSTRACT FROM AUTHOR]

Cardiac tachyarrhythmia presents a significant health care challenge, causing notable morbidity and mortality. Conventional treatments have limitations and potential risks, resulting in an elevated disease burden. Adeno-associated virus (AAV)–mediated gene therapy holds promise as a potential future treatment option. Therefore, we aimed to provide a measured overview of the latest developments in this rapidly growing field. PubMed and Embase databases were searched up to January 2024. Studies that employed AAV as a vector for delivery of therapeutic agents to treat cardiac tachyarrhythmia were included. Of the 26 studies included, 20 published in the last 5 years. There were 22 novel molecular targets identified. More than 80% of the included studies employed small-animal models or used AAV9. In atrial fibrillation preclinical studies, AAV-mediated gene therapy reduced atrial fibrillation inducibility by 81% (odds ratio, 0.19 [0.08–0.45]; P <.01). Similarly, for acquired and inherited ventricular arrhythmia, animal models receiving gene therapy had less inducible ventricular arrhythmia (odds ratio, 0.06 [0.03–0.11]; P <.01). This review highlights the rapid progress of AAV-mediated gene therapy for cardiac tachyarrhythmia. Although these investigations are currently in the early stages of clinical application, they present promising prospects for gene therapy. (PROSPERO registry: CRD42023479448) [ABSTRACT FROM AUTHOR]

Engineering donor organs to better tolerate the harmful non-immunological and immunological responses inherently related to solid organ transplantation would improve transplant outcomes. Our enhanced knowledge of ischemia-reperfusion injury, alloimmune responses and pathological fibroproliferation after organ transplantation, and the advanced toolkit available for gene therapies, have brought this goal closer to clinical reality. Ex vivo organ perfusion has evolved rapidly especially in the field of lung transplantation, where clinicians routinely use ex vivo lung perfusion (EVLP) to confirm the quality of marginal donor lungs before transplantation, enabling safe transplantation of organs originally considered unusable. EVLP would also be an attractive platform to deliver gene therapies, as treatments could be administered to an isolated organ before transplantation, thereby providing a window for sophisticated organ engineering while minimizing off-target effects to the recipient. Here, we review the status of lung transplant first-generation gene therapies that focus on inducing transgene expression in the target cells. We also highlight recent advances in next-generation gene therapies, that enable gene editing and epigenetic engineering, that could be used to permanently change the donor organ genome and to induce widespread transcriptional gene expression modulation in the donor lung. In a future vision, dedicated organ repair and engineering centers will use gene editing and epigenetic engineering, to not only increase the donor organ pool, but to create superior organs that will function better and longer in the recipient. [ABSTRACT FROM AUTHOR]