Viral Vector Gene Research Articles

Polymers as Efficient Non-Viral Gene Delivery Vectors: The Role of the Chemical and Physical Architecture of Macromolecules.

Gene therapy is the technique of inserting foreign genetic elements into host cells to achieve a therapeutic effect. Although gene therapy was initially formulated as a potential remedy for specific genetic problems, it currently offers solutions for many diseases with varying inheritance patterns and acquired diseases. There are two major groups of vectors for gene therapy: viral vector gene therapy and non-viral vector gene therapy. This review examines the role of a macromolecule's chemical and physical architecture in non-viral gene delivery, including their design and synthesis. Polymers can boost circulation, improve delivery, and control cargo release through various methods. The prominent examples discussed include poly-L-lysine, polyethyleneimine, comb polymers, brush polymers, and star polymers, as well as hydrogels and natural polymers and their modifications. While significant progress has been made, challenges still exist in gene stabilization, targeting specificity, and cellular uptake. Overcoming cytotoxicity, improving delivery efficiency, and utilizing natural polymers and hybrid systems are vital factors for prospects. This comprehensive review provides an illuminating overview of the field, guiding the way toward innovative non-viral-based gene delivery solutions.

Targeting Neurological Aspects of Mucopolysaccharidosis Type II: Enzyme Replacement Therapy and Beyond.

Mucopolysaccharidosis type II (MPS II) is a rare, pediatric, neurometabolic disorder due to the lack of activity of the lysosomal hydrolase iduronate 2-sulfatase (IDS), normally degrading heparansulfate and dermatansulfate within cell lysosomes. The deficit of activity is caused by mutations affecting the IDS gene, leading to the pathological accumulation of both glycosaminoglycans in the lysosomal compartment and in the extracellular matrix of most body districts. Although a continuum of clinical phenotypes is described, two main forms are commonly recognized-attenuated and severe-the latter being characterized by an earlier and faster clinical progression and by a progressive impairment of central nervous system (CNS) functions. However, attenuated forms havealso been recently described as presenting some neurological involvement, although less deep, such as deficits of attention and hearing loss. The main treatment for the disease is represented by enzyme replacement therapy (ERT), applied in several countries since 2006, which, albeit showing partial efficacy on some peripheral organs, exhibited a very poor efficacy on bones and heart, and a total inefficacy on CNS impairment, due to the inability of the recombinant enzyme to cross the blood-brain barrier (BBB). Together with ERT, whose design enhancements, performed in the last few years, allowed a possible brain penetration of the drug through the BBB, other therapeutic approaches aimed at targeting CNS involvement in MPS II were proposed and evaluated in the last decades, such as intrathecal ERT, intracerebroventricular ERT, ex vivo gene therapy, or adeno-associated viral vector (AAV) gene therapy. The aim of this review is to summarize the main clinical aspects of MPS II in addition to current therapeutic options, with particular emphasis on the neurological ones and on the main CNS-targeted therapeutic approaches explored through the years.

Adeno-associated viral vector gene therapy: Challenges for the paediatric hepatologist.

Hepatoxicity associated with recombinant adeno-associated virus gene therapy is being increasingly encountered by hepatologists in tertiary and quaternary referral units due to the recent increase of these therapies for neuromuscular and haematological disorders. The challenges in managing the condition stem from a lack of good-quality evidence on the appropriate protocols for immunosuppressants due to lack of representative animal models. There is a need for protocols for diagnosing and treating hepatotoxicity and this possible with further research to understand the problem and its management. The review also highlights the importance of a multidisciplinary team in managing hepatotoxicity and recommends further research to better identify at-risk individuals, define the extent of the problem and assess the long-term effects of liver injury and immunosuppressants.

Widespread correction of brain pathology in feline alpha-mannosidosis by dose escalation of intracisternal AAV vector injection

Alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions in the brain and other tissues. Enzyme replacement therapy is available but is not approved for treating the CNS since the enzyme does not penetrate the blood-brain barrier. However, intellectual disability is a major manifestation of the disease thus a complimentary treatment is needed. While enzyme replacement therapy into the brain is technically feasible, it requires ports and frequent administration over time that are difficult to manage medically. Infusion of adeno-associated viral vectors into the cerebrospinal fluid is an attractive route for broadly targeting brain cells. We demonstrate here the widespread post-symptomatic correction of the globally distributed storage lesions by infusion of a high dose of AAV1-fMANB into the CSF via the cisterna magna in the gyrencephalic alpha-mannosidosis cat brain. Significant improvements in clinical parameters occurred and widespread global correction was documented pre-mortem by non-invasive magnetic resonance imaging. Post-mortem analysis demonstrated high levels of MANB activity and reversal of lysosomal storage lesions throughout the brain. Thus, CSF treatment by adeno-associated viral vector gene therapy appears to be a suitable complement to systemic enzyme replacement therapy to potentially treat the whole patient.

Retention Rates of Genetic Therapies Based on AAV Serotypes 2 and 8 Using Different Drug-Delivery Materials.

The purpose of this study was to compare the retention rate of Adeno-associated viral vector (AAV) gene therapy agents within different subretinal injection systems. The retention of AAV serotype 2-based voretigene neparvovec (VN) and a clinical-grade AAV serotype 8 vector within four different subretinal cannulas from two different manufacturers was quantified. A standardized qPCR using the universal inverted terminal repeats as a target sequence was developed. The instruments compared were the PolyTip® cannula 25 g/38 g by MedOne Surgical, Inc., Sarasota, FL, USA, and three different subretinal injection needles by DORC, Zuidland, The Netherlands (1270.EXT Extendible 41G subretinal injection needle (23G), DORC 1270.06 23G Dual bore injection cannula, DORC 27G Subretinal injection cannula). The retention rate of VN and within the DORC products (10-28%) was comparable to the retention rate (32%) found for the PolyTip® cannula that is mentioned in the FDA-approved prescribing information for VN. For the AAV8 vector, the PolyTip® cannula showed a retention rate of 14%, and a similar retention rate of 3-16% was found for the DORC products (test-retest variability: mean 4.5%, range 2.5-20.2%). As all the instruments tested showed comparable retention rates, they seem to be equally compatible with AAV2- and AAV8-based gene therapy agents.

Development of adenoviral vectors that transduce Purkinje cells and other cerebellar cell-types in the cerebellum of a humanized mouse model

Viral vector gene therapy has immense promise for treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAVs) have had success, their small packaging capacity limits their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, we created chimeric first generation Ad vectors that utilize the hCD46 receptor. Using a "humanized" hCD46 mouse model, we demonstrate these Ad vectors transduce cerebellar cell types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric first generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Current and emerging gene therapies for haemophilia A and B.

After decades of stumbling clinical development, the first gene therapies for haemophilia A and B have been commercialized and have normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several other clinical programs testing adeno-associated viral (AAV) vector gene therapy are at various stages of clinical testing. Multiyear follow-up in phase 1/2 and 3 studies showed long-term and sometimes curative but widely variable and unpredictable efficacy. Liver toxicities, mostly low-grade, occur in the 1st year in at least some individuals in all haemophilia A and B trials and are poorly understood. Wide variability and unpredictability of outcome and slow decline of FVIII levels are a major disadvantage because immune responses to AAV vectors preclude repeat dosing, which otherwise could improve suboptimal or restore declining expression, while overexpression may predispose to thrombosis. Long-term safety outcomes will need lifelong monitoring because AAV vectors infused at high doses integrate into chromosomes at rates that raise questions about potential oncogenicity and necessitate vigilance. Alternative gene transfer systems employing gene editing and/or non-viral vectors are under development and promise to overcome some limitations of the current state of the art for both haemophilia A and B. AAV gene therapies for haemophilia have now become new treatment options but not universal cures. AAV is a powerful but imperfect gene transfer platform. Biobetter FVIII transgenes may help solve some problems plaguing gene therapy for haemophilia A. Addressing variability and unpredictability of efficacy, and delivery of gene therapy to ineligible patient subgroups may require different gene transfer systems, most of which are not ready for clinical translation yet but bring innovations needed to overcome the current limitations of gene therapy.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

AAV gene therapy in companion dogs with severe hemophilia: Real-world long-term data on immunogenicity, efficacy, and quality of life

The hemophilias are the most common severe inherited bleeding disorders and are caused by deficiency of clotting factor (F) VIII (hemophilia A) or FIX (hemophilia B). The resultant bleeding predisposition significantly increases morbidity and mortality. The ability to improve the bleeding phenotype with modest increases in clotting factor levels has enabled the development and regulatory approval of adeno-associated viral (AAV) vector gene therapies for people with hemophilia A and B. The canine hemophilia model has proven to be one of the best predictors of therapeutic response in humans. Here, we report long-term follow-up of 12 companion dogs with severe hemophilia that were treated in a real-world setting with AAV gene therapy. Despite more baseline bleeding than in research dogs, companion dogs demonstrated a 94% decrease in bleeding rates and 61% improvement in quality-of-life over a median of 4.1 years (range 2.6-8.9). No new anti-transgene immune responses were detected; 1 dog with a preexisting anti-FVIII inhibitor achieved immune tolerance with gene therapy. Two dogs expressing 1-5% FVIII post gene therapy experienced fatal bleeding events. These data suggest AAV liver-directed gene therapy is efficacious in a real-world setting, but should target expression > 5% and closely monitor those with levels in the 1-5% range.

Enkephalin Rescues Temporomandibular Joint Pain-Related Behavior in Rats.

Temporomandibular joint disorders include a variety of clinical syndromes that are difficult to manage if associated with debilitating severe jaw pain. Thus, seeking additional experimental therapies for temporomandibular joint pain reduction is warranted. Targeted enkephalin gene therapy approaches provide clear promise for pain control. The studies detailed here indicate significant analgesia and protection of joint tissue are provided after injection of an overexpression viral vector gene therapy near the joint. The viral vector gene therapy described provides overexpression of naturally occurring opioid peptides after its uptake by trigeminal nerve endings. The viral vectors act as independent "minipump" sources for the opioid peptide synthesis in the neuronal cytoplasm producing the intended biological function, reduction of pain, and tissue repair. The antinociceptive effects provided with this delivery method of opioid expression persist for over 4weeks. This is coincident with the expected time frame for the duration of the transgene overproduction of the endogenous opioid peptide before its diminution due to dormancy of the virus. These experimental studies establish a basis for the use of replication-defective herpes simplex type 1-based gene therapy for severe chronic inflammatory temporomandibular joint destruction and pain. As innovative means of significantly reducing joint inflammation and preserving tissue architecture, gene therapies may extend their clinical usefulness for patients with temporomandibular joint disorders.

Interspecies Scaling of Transgene Products for Viral Vector Gene Therapies: Method Assessment Using Data from Eleven Viral Vectors.

The prediction of transgene product expression in human is important to guide first-in-human (FIH) dose selection for viral vector-based gene replacement therapies. Recently, allometric scaling from preclinical data and interspecies normalization of dose-response (D-R) relationship have been used to predict human transgene product expression of adeno-associated virus (AAV) vectors. In this study, we assessed two interspecies allometric scaling methods and two dose-response methods in predicting human transgene product expression of nine intravenously administered AAV vectors, one intramuscularly administered AAV vector, and one intravesical administered adenoviral vector. Among the four methods, normalized D-R method generated the highest prediction accuracy, with geometric mean fold error (GMFE) of 2.9 folds and 75% predictions within fivefold deviations of observed human transgene product levels. The vg/kg-based D-R method worked well for locally delivered vectors but substantially overpredicted human transgene product levels of some hemophilia A and B vectors. For both intravenously and locally administered vectors, the prediction accuracy of allometric scaling using body weight^-0.25 (AS by W^-0.25) was superior to allometric scaling using log(body weight) (AS by logW). This study successfully extended the use of allometric scaling and interspecies D-R normalization methods for human transgene product prediction from intravenous viral vectors to locally delivered viral vectors.

Molecular camouflage by a context-specific hydrogel as the key to unlock the potential of viral vector gene therapy

Gene therapy offers hope for currently untreatable diseases; the patient’s own cellular machinery is recruited to create therapeutics. However, unpredictable responses that lead to neutralization by the host immune system and issues in constraining, controlling and sustaining delivery have presented clinical barriers to otherwise promising therapeutic developments. Here, we show that the protective environment provided by advanced biomaterials can function as injectable gene carriers to focus their therapeutic potential. Firstly, we investigated the potential of a tissue-specific molecular hydrogel to package recombinant adeno-associated viruses (rAAVs). Once a delivery pathway was confirmed, a set of rAAV variants were subsequently assessed for their ability to transduce various types of rodent and human neural cells in vitro and in vivo. Based on GFP expression, we identified a relatively new variant, rAAV-DJ, as showing desirable characteristics for constrained delivery and transduction efficiency. For the first time, we demonstrated precise control over the strength and type of interaction between biomaterials and rAAVs enabling the programmed release of viral payloads. This new approach enables specific infection of desired anatomical targets in a programmed fashion.

CRISPR-Cas9 KO Cell Line Generation and Development of a Cell-Based Potency Assay for rAAV-FKRP Gene Therapy.

Limb-Girdle Muscular Dystrophy R9 (LGMDR9) is a dystroglycanopathy caused by Fukutin-related protein (FKRP) defects leading to the deficiency of α-DG glycosylation, essential to membrane integrity. Recombinant adeno-associated viral vector (rAAV) gene therapy offers great therapeutic promise for such neuromuscular disorders. Pre-clinical studies have paved the way for a phase 1/2 clinical trial aiming to evaluate the safety and efficacy of FKRP gene therapy in LGMDR9 patients. To demonstrate product activity, quality, and consistency throughout product and clinical development, regulatory authorities request several quality controls, including a potency assay aiming to demonstrate and quantify the intended biological effect of the gene therapy product. In the present study, we generated FKRP knock-out (KO) cells fully depleted of α-DG glycosylation using CRISPR-Cas9 to assess the functional activity of a rAAV-FKRP gene therapy. We then developed a high-throughput On-Cell-Western methodology to evaluate the restoration of α-DG glycosylation in KO-FKRP cells and determine the biological activity of the FKRP transgene. The determination of the half maximal effective concentration (EC50) provides a method to compare the rAAV-FKRP batch using a reference standard. The generation of KO-FKRP muscle cells associated with the high-throughput On-Cell-Western technique may serve as a cell-based potency assay to assess rAAV-FKRP gene therapy products.

Potential of Adeno-associated virus Vector Gene Therapy for Achromatopsia

Achromatopsia atau yang dikenal dengan buta warna adalah kurangnya kemampuan untuk membedakan warna-warna tertentu. Dimana hal ini merupakan ketidakmampuan sel-sel kerucut mata untuk menangkap suatu spektrum warna tertentu akibat faktorgenetik dan penyakit tertentu. Kelainan pada struktur anatomi mata yang terletak pada retina dan diturunkan secara resesif.Mutasi ini menyebabkan hilangnya fungsional dan degenerasi progresif lambat dari fotoreseptor sel kerucut. Terapi padabuta warna saat ini hanya terbatas pada konseling genetik, kacamata, atau lensa kontak berwarna. Metode terapi genetiksaat ini sedang dikembangkan dengan menargetkan organ retina dengan menggunakan vektor rekombinan adenovirus. Hasil penelitian yang dilakukan pada hewan uji ditemukan adanya perbaikan fungsi pada sel kerucut sehingga terapi gen merupakan modalitas yang cukup menjanjikan di masa mendatang.

Recent insights into lysosomal acid lipase deficiency

Lysosomal acid lipase (LAL) is the sole enzyme known to degrade neutral lipids in the lysosome. Mutations in the LAL-encoding LIPA gene lead to rare lysosomal lipid storage disorders with complete or partial absence of LAL activity. This review discusses the consequences of defective LAL-mediated lipid hydrolysis on cellular lipid homeostasis, epidemiology, and clinical presentation. Early detection of LAL deficiency (LAL-D) is essential for disease management and survival. LAL-D must be considered in patients with dyslipidemia and elevated aminotransferase concentrations of unknown etiology. Enzyme replacement therapy, sometimes in combination with hematopoietic stem cell transplantation (HSCT), is currently the only therapy for LAL-D. New technologies based on mRNA and viral vector gene transfer are recent efforts to provide other effective therapeutic strategies.

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri‑lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri‑lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

Visualization research on ENT1/NIS dual-function gene therapy to reverse drug resistance mediated by MUC1 in GEM-resistant pancreatic cancer

PurposeTo use bifunctional target genes to increase the intracellular transport of gemcitabine (GEM) to reverse chemotherapy resistance and to simultaneously use reporter gene imaging to localize therapeutic genes. The therapeutic effect was evaluated by [18F]FLT PET/CT to visualize the effect of gene therapy. MethodsA viral gene vector containing the pancreatic cancer-targeting promoter MUC1 for specific transcription of equilibrative nucleoside transporter 1 (ENT1) and NIS (nuclide transport channel) was employed. [125I]NaI uptake tests and [131I]NaI SPECT imaging were performed to verify the function of NIS and the target function of MUC1. The correlation between [18F]FLT uptake and GEM resistance were assessed, and the influence ENT1 and thymidine kinase 1 (TK1) expression on [18F]FLT micro-PET/CT was measured, which provides a theoretical basis for the use of [18F]FLT micro-PET/CT to evaluate the efficacy of gene therapy. ResultsFirst, functions of gene therapy were confirmed: ENT1 reversed the drug resistance of GEM-resistant pancreatic cancer cells by increasing GEM intracellular transport; MUC1 drove NIS target gene expression in pancreatic cancer; and therapeutic genes could be localized using [131I]NaI SPECT reporter gene imaging. Second, the [18F]FLT uptake ratio was affected by drug resistance and GEM treatment. The mechanism underlying this effect was related to ENT1 and TK1. Increased expression of ENT1 inhibited the expression of TK1 after GEM chemotherapy to reduce the uptake of [18F]FLT. Finally, micro-PET/CT indicated that the SUVmax of [18F]FLT could predict survival time. SUVmax exhibited an increasing trend in resistant pancreatic cancer but a trend of inhibition after upregulation of ENT1, which was more significant after GEM treatment. ConclusionsBifunctional targeted genes can localize therapeutic genes through reporter gene imaging, reverse the drug resistance of GEM-resistant pancreatic cancer and be visually evaluated through [18F]FLT micro-PET/CT.

Evaluation of Cellular Immune Response to Adeno-Associated Virus-Based Gene Therapy.

The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment.

Baseline Characteristics and Interim Safety in RESPOND: A Phase 4 Study in Children with Spinal Muscular Atrophy (SMA) Treated With Nusinersen After Onasemnogene Abeparvovec (P7-9.003)

Baseline Characteristics and Interim Safety in RESPOND: A Phase 4 Study in Children with Spinal Muscular Atrophy (SMA) Treated With Nusinersen After Onasemnogene Abeparvovec (P7-9.003)

Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.