Clinical Success Of Gene Therapy Research Articles

Gene therapy for inherited retinal diseases: exploiting new tools in genome editing and nanotechnology.

Inherited retinal diseases (IRDs) encompass a diverse group of genetic disorders that lead to progressive visual impairment and blindness. Over the years, considerable strides have been made in understanding the underlying molecular mechanisms of IRDs, laying the foundation for novel therapeutic interventions. Gene therapy has emerged as a compelling approach for treating IRDs, with notable advancements achieved through targeted gene augmentation. However, several setbacks and limitations persist, hindering the widespread clinical success of gene therapy for IRDs. One promising avenue of research is the development of new genome editing tools. Cutting-edge technologies such as CRISPR-Cas9 nucleases, base editing and prime editing provide unprecedented precision and efficiency in targeted gene manipulation, offering the potential to overcome existing challenges in gene therapy for IRDs. Furthermore, traditional gene therapy encounters a significant challenge due to immune responses to viral vectors, which remain crucial obstacles in achieving long-lasting therapeutic effects. Nanotechnology has emerged as a valuable ally in the quest to optimize gene therapy outcomes for ocular diseases. Nanoparticles engineered with nanoscale precision offer improved gene delivery to specific retinal cells, allowing for enhanced targeting and reduced immunogenicity. In this review, we discuss recent advancements in gene therapy for IRDs and explore the setbacks that have been encountered in clinical trials. We highlight the technological advances in genome editing for the treatment of IRDs and how integrating nanotechnology into gene delivery strategies could enhance the safety and efficacy of gene therapy, ultimately offering hope for patients with IRDs and potentially paving the way for similar advancements in other ocular disorders.

Frequency and phenotypic characteristics of RPE65 mutations in the Chinese population

BackgroundThe retinoid isomerohydrolase RPE65 has received considerable attention worldwide since a successful clinical gene therapy was approved in 2017 as the first treatment for vision loss associated with RPE65-mediated inherited retinal disease. Identifying patients with RPE65 mutations is a prerequisite to assessing the patients’ eligibility to receive RPE65-targeted gene therapies, and it is necessary to identify individuals who are most likely to benefit from gene therapies. This study aimed to investigate the RPE65 mutations frequency in the Chinese population and to determine the genetic and clinical characteristics of these patients.ResultsOnly 20 patients with RPE65 mutations were identified, and RPE65 mutations were determined to be the 14th most common among all patients with genetic diagnoses. Ten novel variants and two hotspots associated with FAP were identified. A literature review revealed that a total of 57 patients of Chinese origin were identified with pathogenic mutations in the RPE65 gene. The mean best Snellen corrected visual acuity was worse (mean 1.3 ± 1.3 LogMAR) in patients older than 20 years old than in those younger than 15 years old (0.68 ± 0.92 LogMAR). Bone spicule-like pigment deposits (BSLPs) were observed in six patients; they were older than those without BSLP and those with white-yellow dots. Genotype–phenotype analysis revealed that truncating variants seem to lead to a more severe clinical presentation, while best corrected visual acuity testing and fundus changes did not correlate with specific RPE65 variants or mutation types.ConclusionsThis study provides a detailed clinical-genetic assessment of patients with RPE65 mutations of Chinese origin. These results may help to elucidate RPE65 mutations in the Chinese population and may facilitate genetic counseling and the implementation of gene therapy in China.

Human Lentiviral Gene Therapy Restores the Cellular Phenotype of Autosomal Recessive Complete IFN-γR1 Deficiency

Autosomal recessive (AR) complete interferon-γ receptor 1 (IFN-γR1) deficiency, also known as one genetic etiology of Mendelian susceptibility to mycobacterial disease (MSMD), is a life-threatening congenital disease leading to premature death. Affected patients present a pathognomonic predisposition to recurrent and severe infections with environmental mycobacteria or the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. Current therapeutic options are limited to antibiotic treatment and hematopoietic stem cell transplantation, however with poor outcome. Given the clinical success of gene therapy, we introduce the first lentiviral-based gene therapy approach to restore expression and function of the human IFN-γR-downstream signaling cascade. In our study, we developed lentiviral vectors constitutively expressing the human IFN-γR1 and demonstrate stable transgene expression without interference with cell viability and proliferation in transduced human hematopoietic cells. Using an IFN-γR1-deficient HeLa cell model, we show stable receptor reconstitution and restored IFN-γR1 signaling without adverse effect on cell functionality. Transduction of both SV40-immortalized and primary fibroblasts derived from IFN-γR1-deficient MSMD patients was able to recover IFN-γR1 expression and restore type II IFN signaling upon stimulation with IFN-γ. In summary, we highlight lentiviral vectors to correct the IFN-γ mediated immunity and present the first gene therapy approach for patients suffering from AR complete IFN-γR1 deficiency.

Peptide-based targeted polymeric nanoparticles for siRNA delivery

The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.

RPE65 mutation frequency and phenotypic variation according to exome sequencing in a tertiary centre for genetic eye diseases in China.

PurposeRetinoid isomerohydrolase RPE65 has received a tremendous amount of attention due to successful clinical gene therapy for Leber congenital amaurosis (LCA) cases caused by RPE65 mutations. This study aimed to evaluate the frequency of RPE65 mutations and the associated phenotypes based on exome sequencing.Methods RPE65 variants were collected from exome sequencing data obtained from 2133 probands with different forms of hereditary retinal degeneration (HRD). Clinical data were collected from probands with homozygous or compound heterozygous variants in RPE65. Associated phenotypes were characterized based on clinical data.ResultsBiallelic RPE65 mutations were detected in 18 families, including eight with LCA, five with early‐onset retinal degeneration, four with fundus albipunctatus‐like (FA‐like) changes and one with high hyperopia. These cases accounted for approximately 3.0% (8/269) of LCA and 0.8% (18/2133) of HRD cases. An almost identical FA‐like change was identified in seven patients from four unrelated families with RPE65 mutations. Classification of mutations suggested that FA‐like changes may be associated with biallelic missense mutations in RPE65.ConclusionFundus albipunctatus‐like (FA‐like) change, a common characteristic fundus sign in RPE65 biallelic mutations, was unexpected but was confirmed by the finding that affected siblings from different families exhibited similar phenotypes. These results enrich our understanding of RPE65 mutation frequencies and their associated phenotypic variants.

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras.

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

Synthesis, characterisation and preliminary investigation of the haemocompatibility of polyethyleneimine-grafted carboxymethyl chitosan for gene delivery

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. In the present study, carboxymethyl chitosan (CMCS) was prepared by chitosan (CS) alkalisation and carboxymethylation reactions. Then polyethyleneimine (PEI) was grafted to the backbone of CMCS by an amidation reaction. The CMCS–PEI copolymer showed strong complexation capability with DNA to form nanoparticles, and achieved lower cytotoxicity and higher transfection efficiency compared with PEI (25kDa) towards 293T and 3T3 cells. Moreover, the haemocompatibility of the CMCS–PEI copolymer was investigated through the aggregation, morphology and lysis of human red blood cells (RBCs), along with the impact on the clotting function with activated partial thromboplastin time (APTT), prothrombin time (PT) and thromboelastographic (TEG) assays. The results demonstrated that the CMCS–PEI copolymer with a concentration lower than 0.05mg/mL had little impact on the aggregation, morphology or lysis of RBCs, or on blood coagulation. Therefore, the copolymer may be a strong alternative candidate as an effective and safe non-viral vector.

Engineered AAV vectors for improved central nervous system gene delivery.

Adeno-associated viruses (AAV) are non-pathogenic members of the Parvoviridae family that are being harnessed as delivery vehicles for both basic research and increasingly successful clinical gene therapy. To address a number of delivery shortcomings with natural AAV variants, we have developed and implemented directed evolution—a high-throughput molecular engineering approach to generate novel biomolecules with enhanced function—to create novel AAV vectors that are designed to preferentially transduce specific cell types in the central nervous system (CNS), including astrocytes, neural stem cells, and cells within the retina. These novel AAV vectors—which have enhanced infectivity in vitro and enhanced infectivity and selectivity in vivo—can enable more efficient studies to further our understanding of neurogenesis, development, aging, and disease. Furthermore, such engineered vectors may aid gene or cell replacement therapies to treat neurodegenerative disease or injury.

ET-38 * BRAIN PENETRATING NON-VIRAL GENE VECTORS FOR EFFICIENT GENE TRANSFER TO BRAIN TUMORS

Gene therapy constitutes an attractive approach for curing a variety of CNS diseases, including glioblastoma. Multiple studies have been carried out focusing on improving the total level and distribution of gene expression throughout the brain tissue. Viral gene delivery, though relatively efficient, has been limited by immunogenicity, low packaging capacity and difficulties in scale-up. Non-viral gene vectors offer an alternative strategy but are limited in their ability to generate high-level, widespread therapeutic effect. The nanoporous and electrostatically charged extracellular space found between healthy or tumor cells constitutes a steric and adhesive barrier, which hampers widespread distribution of gene vectors in the brain, regardless of administration method, thereby limiting gene transfer to target cells [1]. Indeed, following convention enhanced delivery (CED) conventional gene vectors are confined to the point of administration, limiting the clinical success of gene therapy for glioblastoma [2]. We developed a strategy to formulate small, stable and densely coated cationic polymer-based gene vectors that have the physicochemical characteristics required to overcome this ‘brain tissue barrier’. Using high resolution multiple particle tracking, we assessed the diffusion rates of gene vectors in freshly extracted ex vivo healthy and tumor rodent brain tissues. We thoroughly characterized their toxicity, cell uptake and transfection efficacy in vitro. Finally, we analyzed the ability of these vectors to rapidly penetrate and distribute throughout the brain tissue in vivo, thus reaching and transfecting cells over a large volume following CED. 1.Nance, E.A., et al., Sci Transl Med, 2012. 2.Voges, J., et al., Ann Neurol, 2003.

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

High-Throughput Methods for Screening Polymeric Transfection Reagents

The clinical success of gene therapy requires the development of a safe and efficient delivery system for DNA. Cationic polymers are nonviral vectors that can associate electrostatically with plasmid DNA to form nanocomplexes. In some cases, this is sufficient for cellular uptake and transfection, although the precise mechanisms by which polymers facilitate gene delivery remain unclear. A robust and reliable method to screen for efficacy is essential for the development of effective polycationic transfection reagents. Numerous parameters must be controlled and optimized, such as polymer structure, polymer-DNA-binding conditions (mixing method, pH, ionic strength, incubation time, concentrations, ratios), transfection media (type, serum content), DNA dose and incubation time with the cells, cell specificity, and assay conditions. In this protocol, we describe a high-throughput method for assessing polymer-mediated transfection. The technique uses 96-well plates, which allows many transfection parameters to be varied and optimized in parallel. Hundreds of polymers can be tested in quadruplicate in a single day and the technique can easily be automated to efficiently and reproducibly test large material libraries. One limitation is that many plate types, solutions, and equipment must be stocked and sterilized. Moreover, because all polymers are processed simultaneously in very small volumes, it is difficult to validate each step for each polymer to ensure solution uniformity and adequate polymer-DNA complexation. Despite these drawbacks, this high-throughput screening method has already been used successfully in the development of efficient polycation vectors.

Spermine‐alt‐poly(ethylene glycol) polyspermine as a safe and efficient aerosol gene carrier for lung cancer therapy

The clinical success of gene therapy critically depends upon the safety and efficiency of delivery system used. Although polyethylenimine (PEI) has been commonly used as an efficient cationic polymeric gene carrier due to its high transfection efficiency, its cytotoxicity and nondegradability limit the polymer's therapeutic applications in clinical trials. In this study, biocompatible polyspermine based on spermine (SPE) and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) was synthesized using a Michael-type addition reaction, and its ability as an alternative gene carrier for lung cancer therapy was evaluated. SPE-alt-PEG polyspermine was complexed with plasmid DNA, and the resulting complexes were characterized by particle size and surface charge by dynamic light scattering, complex formation and DNA protection ability by gel retardation, and complex shape by energy-filtering transmission electron microscopy. The SPE-alt-PEG copolymer showed low cytotoxicity, and SPE-alt-PEG/DNA complexes showed efficacious transfection efficiency compared with 25 kDa PEI (PEI 25K). Also SPE-alt-PEG/GFP complexes were efficiently transferred into the lungs after aerosol administration without toxicity, and delivery of Pdcd4 gene as a therapeutic gene with SPE-alt-PEG polyspermine greatly reduced tumor size as well as tumor numbers in K-ras(LA1) lung cancer model mice compared relative to the effect observed for PEI 25K. These results suggest that SPE-alt-PEG has potential as a gene carrier for lung cancer gene therapy.

Cyclodextrin-scaffolded glycotransporters for gene delivery

Conventional drugs consist of a formulation of a bioactive species and a carrier, the former accounting for most of the sophistication of the design. In the case of biomolecular drugs, however, the role of the carrier becomes decisive in enabling the load to reach its target to carry out its designed therapeutic function. Thus, the clinical success of gene therapy, where the active principles are nucleic acids, critically depends on the use of efficient and safe delivery systems. Carbohydrates have proven particularly useful in this regard. Glycocoating, similarly to poly(ethylene)glycol (PEG)-coating (pegylation), can stabilize colloidal aggregates by improving solvation and preventing nonspecific interactions, for example, with serum proteins. Moreover, glycoconjugates can drive specific recognition and receptor-mediated internalization in target cells. Actually, the inherent flexibility of carbohydrate and glycoconjugate chemistry has greatly contributed to enlarging the range of functional materials that can be rationally conceived for gene delivery. Herein, this is illustrated with selected examples that focus on controlling the architectural parameters of the vectors to make them suitable for structure–activity relationship (SAR) and optimization studies. The members of the cyclomaltooligosaccharide (cyclodextrin, CD) family will be the central actors of the story.

Lipoic Acid Modified Low Molecular Weight Polyethylenimine Mediates Nontoxic and Highly Potent in Vitro Gene Transfection

The clinical success of gene therapy intimately relies on the development of safe and efficient gene carrier systems. We found here that 1.8 kDa polyethylenimine (PEI) following hydrophobic modification with lipoic acid (LA) mediated nontoxic and highly potent in vitro gene transfection in both HeLa and 293T cells. 1.8 kDa PEI-LA conjugates were prepared with controlled degree of substitution (DS) by coupling LA to PEI using carbodiimide chemistry. Gel electrophoresis measurements showed that the DNA binding ability of 1.8 kDa PEI was impaired by lipoylation, in which an N/P ratio of 2/1 and 4-6/1 was required for 1.8 kDa PEI and 1.8 kDa PEI-LA conjugates, respectively, to completely inhibit DNA migration. Interestingly, dynamic light scattering measurements (DLS) revealed that PEI-LA conjugates condensed DNA into much smaller sizes (183-84 nm) than unmodified 1.8 kDa PEI (444-139 nm) at N/P ratios ranging from 20/1 to 60/1. These polyplexes revealed similar surface charges of ca. +22 to +30 mV. 1.8 kDa PEI-LA(2) polyplexes formed at an N/P ratio of 10/1 were stable against exchange with 12-fold excess of negatively charged dextran sodium sulfate (DSS) relative to DNA phosphate groups while 1.8 kDa PEI controls dissociated at 6-fold excess of DSS, indicating that lipoylation of 1.8 kDa PEI resulted in stronger binding with DNA. Importantly, DNA was released from 1.8 kDa PEI-LA(2) polyplexes upon addition of 10 mM dithiothreitol (DTT). Reduction-triggered unpacking of 1.8 kDa PEI-LA(2) polyplexes was also confirmed by DLS. MTT assays demonstrated that all PEI-LA conjugates and polyplexes were essentially nontoxic to HeLa and 293T cells up to a tested concentration of 50 μg/mL and an N/P ratio of 80/1, respectively. The in vitro gene transfection studies in HeLa and 293T cells showed that lipoylation of 1.8 kDa PEI markedly boosted its transfection activity. For example, 1.8 kDa PEI-LA(2) polyplexes displayed 400-fold and 500-fold higher levels of gene expression than unmodified 1.8 kDa PEI controls, which were ca. 2-fold and 3-fold higher than 25 kDa PEI controls, in serum-free and 10% serum media, respectively. The transfection efficiency decreased with increasing DS, following an order of 1.8 kDa PEI-LA(2) > 1.8 kDa PEI-LA(4) > 1.8 kDa PEI-LA(6) ≫ 1.8 kDa PEI. Confocal laser scanning microscopy (CLSM) studies corroborated that 1.8 kDa PEI-LA(2) delivered and released DNA into the nuclei of HeLa cells more efficiently than 25 kDa PEI. These nontoxic 1.8 kDa PEI-LA conjugates form a superb basis for the development of targeting, biocompatible and highly efficient carriers of gene delivery.

Novel hyaluronic acid–chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis

Gene therapy is a promising new treatment strategy for common joint-disorders such as osteoarthritis. The development of safe, effective, targeted non-viral gene carriers is important for the clinical success of gene therapy. The present work describes the use of hybrid hyaluronic acid (HA)/chitosan (CS) nanoparticles as novel non-viral gene delivery vectors capable of transferring exogenous genes into primary chondrocytes for the treatment of joint diseases. HA/CS plasmid–DNA nanoparticles were synthesized through the complex coacervation of the cationic polymers with pEGFP. Particle size and zeta potential were related to the weight ratio of CS to HA, where increases in nanoparticle size and decreases in surface charge were observed as HA content increased. The particle size and the zeta potential varied according to pH. Transfection of primary chondrocytes was performed under different conditions to examine variations in the pH of the transfection medium, different N/P ratios, different plasmid concentrations, and different molecular weights of chitosan. Transfection efficiency was maximized for a medium pH of approximately 6.8, an N/P ratio of 5, plasmid concentration of 4 μg/ml, and a chitosan molecular weight of 50 kDa. The transfection efficiency of HA/CS-plasmid nanoparticles was significantly higher than that of CS-plasmid nanoparticles under the same conditions. The average viability of cells transfected with HA/CS-plasmid nanoparticles was over 90%. These results suggest that HA/CS-plasmid nanoparticles could be an effective non-viral vector suitable for gene delivery to chondrocytes.

A novel cationic liposome formulation for efficient gene delivery via a pulmonaryroute

The clinical success of gene therapy for lung cancer is not only dependent on efficient genecarriers but also on a suitable delivery route. A pulmonary delivery route can directlydeliver gene vectors to the lung which is more efficient than a systemic delivery route. Forgene carriers, cationic liposomes have recently emerged as leading non-viral vectors inworldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are oftenobserved which is mostly dependent on the cationic lipid used. Therefore, an efficient andsafe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our groupwas first used to prepare cationic liposomes. Physicochemical and biological properties ofLHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positivesurface charge, spherical morphology, a relatively narrow particle size distributionand strong DNA binding capability. Compared with Lipofectamine2000, the newcationic liposome formulation using LHLN exhibited not only lower cytotoxicity(P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cellsfor in vitro tests. When administered by intratracheal instillation into ratlungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higherpulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficientpulmonary gene delivery.

Cationic amphiphiles: promising carriers of genetic materials in gene therapy

The clinical success of gene therapy critically depends on the use of efficient and safe gene delivery reagents. The present tutorial review is aimed at inspiring young researchers and students to take up the unsolved challenges in using cationic amphiphiles as safe gene transfer reagents. The review highlights important structure-activity studies in the field to date including the use of cationic amphiphiles for receptor specific targeted gene therapy and for delivery of siRNAs in the emerging field of RNA interference.

Muscular Gene Transfer Using Nonviral Vectors

Skeletal muscle is a target tissue of choice for the gene therapy of both muscle and non-muscle disorders. Investigations of gene transfer into muscle have progressed considerably from the expression of plasmid reporter genes to the production of therapeutic proteins such as trophic factors, hormones, antigens, ion channels or cytoskeletal proteins. Viral vectors are intrinsically the most efficient vehicles to deliver genes into skeletal muscles. But, because viruses are associated with a variety of problems (such as immune and inflammatory responses, toxicity, limited large scale production yields, limitations in the size of the carried therapeutic genes), nonviral vectors remain a viable alternative. In addition, as nonviral vectors allow to transfer genetic structures of various sizes (including large plasmid DNA carrying full-length coding sequences of the gene of interest), they can be used in various gene therapy approaches. However, given the lack of efficiency of nonviral vectors in experimental studies and in the clinical settings, the overall outcome clearly indicates that improved synthetic vectors and/or delivery techniques are required for successful clinical gene therapy. Today, most of the potential muscle-targeted clinical applications seem geared toward peripheral ischemia (mainly through local injections) and cancer and infectious vaccines, and one locoregional administration of naked DNA in Duchenne muscular dystrophy. This review updates the developments in clinical applications of the various plasmid-based non-viral methods under investigation for the delivery of genes to muscles.

The Clonal Inventory of Gene Corrected Hematopoiesis in Three Successful Clinical Gene Therapy Trials

To enhance the safety and efficacy of future gene therapy trials using integrating vector systems it is necessary to analyse the clonality of the genetically modified cell pool. The comparative analyses of integration site distribution and cellular gene expression will further reveal causal mechanisms of in vivo clone selection. We followed the repopulation clonality of 21 patients which participated in 3 successful clinical gene therapy trials via linear amplification mediated PCR (LAM PCR). We identified the integration sites (IS) of pre and post transplantation samples by Sanger sequencing and accomplished RNA analyses. The comparative results from all trials showed that vector integration is favoured in gene coding regions, in particular transcriptional start sites. In both X-SCID trials significantly more post transplantation IS were located in or in the vicinity of genes encoding proteins with kinase or transferase activity. In pre transplantation samples no uniform gene class was overrepresented. In both trials we detected common insertion sites mainly post transplantation and the effect was more pronounced in the trial where 4 patients developed vector induced leukemia. Notably, we detected no significant differences regarding the IS distribution in leukemic versus non leukemic patients. The gene corrected repopulation of patient 1 and 2 of the X-CGD trial was polyclonal until 542 and 777 days after transplantation, respectively. 5 months after therapy dominant clones appeared. In patient 1, between 616 and 820 days post transplantation (post mortem time point) the number of participating clones and the contribution of a dominant clone decreased while the contribution of another dominant clone increased. In both patients the integrated vector induced the upregulation of the genes MDS1/EVI1, PRDM16 or SETBP1 and thus led to the in vivo expansion of affected cell clones. From these trials we sequenced >2000 unique IS by Sanger sequencing and several thousand via pyrosequencing (datamining is ongoing). Our data show that the integration site distribution was non random, that the integrated vector influenced the cellular gene expression which caused subtle to massive changes in the repopulation clonality and that it will soon be possible to define the clonal inventory of patients using next generation sequencing technologies.

Cationic liposomes as non‐viral carriers of gene medicines: Resolved issues, open questions, and future promises

The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as "transfection vectors." The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantages associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling and preparation techniques; (c) ability to inject large lipid:DNA complexes; and (d) low immunogenic response. The present review highlights the major achievements in the area of designing efficacious cationic transfection lipids, some of the more recent advances in the field of cationic liposomes-mediated gene transfer and targeted gene delivery, some unresolved issues and challenges in liposomal gene delivery, and future promises of cationic liposomes as gene-carriers in non-viral gene therapy.