Gene Therapy Of Human Diseases Research Articles

The RRE-Rev Module Has No Effect on the Packaging Efficiency of Cas9 and Gag Proteins into NanoMEDIC Virus-like Particles.

Delivery of ribonucleoprotein complexes of Cas9 nuclease and guide RNA into target cells with virus-like particles (VLP) is one of the novel methods of genome editing and is suitable for gene therapy of human diseases in the future. The efficiency of genome editing with VLPs depends on the Cas9 packaging into VLPs, the process mediated by the viral Gag protein. To improve the packaging of Cas9 into NanoMEDIC VLPs, plasmid constructs for Cas9 and Gag expression were modified by adding the HIV Rev response element (RRE), which was expected to increase the nuclear export of RRE-containing transcripts into the cytosol via the Rev accessory protein, as described for a Vpr-Cas9-based VLP system. The Cas9 and Gag protein levels in cell lysates were found to increase upon cotransfection with either the Rev-expressing plasmid or the empty control plasmid. The effect was independent of the presence of RRE in the transcript. Moreover, AP21967-induced dimerization of FRB and FKBP12, but not plasmid modification with RRE and/or cotransfection with the Rev-expressing plasmid, was shown to play the major role in Cas9 packaging into NanoMEDIC VLPs. The data indicated that it is impractical to use the RRE-Rev module to enhance the packaging of Cas9 nuclease into VLPs.

Recognizing CRISPR as the new age disease-modifying drug: Strategies to bioengineer CRISPR/Cas for direct in vivo delivery.

Clustered regularly interspaced short palindromic repeats (CRISPR) have established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g., lentivirus) and non-viral encapsulation (e.g., lipid particles, polymer-based, and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated.

Gene Therapy for Human Diseases: Recent Achievements and Near-Term Development Prospects

The article briefly summarizes recent advances in genetic medicine that paved the way for the further development of gene therapy and set the stage for the development of next generation technology. Issues related to the main obstacle for wider application of gene therapy methods, in particular, with the immune response to gene delivery vectors and transgene products are considered. In this context, the role of new technology allowing to bypass the immune obstacle, such as development of modified capsids of adeno-associated viruses (AAV) and methods for temporary removal of antibodies from the bloodstream, as well as gene transfer into tissues using nanoparticles, is discussed. Along with the technology of the first generation gene therapy focused on the delivery of transgenes into target tissues, latest advances in the development of a completely new approach to gene therapy which is based on precise modification of the human genome sequence, gene editing technology, are summarized. Finally, promising next-generation gene editing technology is outlined, such as RNA-targeted editing technology and epigenome editing technology, which are more specific, precise, efficient and applicable to different groups of diseases. The article concludes that gene therapy and, in particular, human genome editing is perhaps the most exciting and revolutionary biotechnology of our time, due to both recent developments and opportunities it might provide in the nearest future.

Knockout of the CMP-Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays.

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP–sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.

CRISPR-Directed Therapeutic Correction at the NCF1 Locus Is Challenged by Frequent Incidence of Chromosomal Deletions

Resurrection of non-processed pseudogenes may increase the efficacy of therapeutic gene editing, upon simultaneous targeting of a mutated gene and its highly homologous pseudogenes. To investigate the potency of this approach for clinical gene therapy of human diseases, we corrected a pseudogene-associated disorder, the immunodeficiency p47phox-deficient chronic granulomatous disease (p47phox CGD), using clustered regularly interspaced short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) to target mutated neutrophil cytosolic factor 1 (NCF1). Being separated by less than two million base pairs, NCF1 and two pseudogenes are closely co-localized on chromosome 7. In healthy people, a two-nucleotide GT deletion (ΔGT) is present in the NCF1B and NCF1C pseudogenes only. In the majority of patients with p47phox CGD, the NCF1 gene is inactivated due to a ΔGT transfer from one of the two non-processed pseudogenes. Here we demonstrate that concurrent targeting and correction of mutated NCF1 and its pseudogenes results in therapeutic CGD phenotype correction, but also causes potentially harmful chromosomal deletions between the targeted loci in a p47phox-deficient CGD cell line model. Therefore, development of genome-editing-based treatment of pseudogene-related disorders mandates thorough safety examination, as well as technological advances, limiting concurrent induction of multiple double-strand breaks on a single chromosome.

THE DEVELOPMENT of a SAFE and OPTIMIZED GENE THERAPY for HUMAN DISEASES

Gene therapy is the therapeutic delivery of a gene or nucleic acid into a patient’s cells to cure or alleviate the symptoms of a disease that was caused by genetic malfunction, either gain- of loss-of function. Throughout the years, gene therapy has been faced with fluctuations of development before reaching its current stage. In the early stage, gene therapy was concerned to possess several problems such as toxicity, mutagenesis, and adverse immune responses which would harm the patients, instead of benefiting them. Fortunately, gene therapy has currently reached the phase where its administration can be performed in a safe, controllable manner with a good tolerability and excellent therapeutic effect. This review will recite the development of gene therapy research, highlight the vector-related safety issues, and discuss the latest updates in recent clinical trials with promising results in correcting gene defects in the cell, reducing the symptoms of the disease, as well as improving the patient’s quality of life.

Modified Exosomes Reduce Apoptosis and Ameliorate Neural Deficits Induced by Traumatic Brain Injury.

Apoptosis contributes to the pathogenesis of traumatic brain injury (TBI). Engineered exosomes incorporated with therapeutic nuclear acids have been explored for gene therapy for human diseases. The current study sought to investigate the effect of modified exosome-containing plasmids expressing B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X-protein (Bax) short hairpin RNA (shRNA) on apoptosis and neural functions after TBI. C57BL/6J mice were subjected to controlled cortical impact injury and were treated with the modified exosomes. The results showed that modified exosomes attenuated the decrease of myeloid cell leukemia-1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), and Survivin protein levels in the brain and reduced Cytochrome c release from mitochondria to cytosol after TBI. They also attenuated the impairments of miniature excitatory postsynaptic current (mEPSC) and long-term potentiation (LTP) in the hippocampus of TBI mice and improved the motor and cognitive behaviors after TBI. These results suggested that the modified exosomes might reduce apoptosis and ameliorate neural and functional deficits in mouse models of TBI.

Highly branched polymers deliver

Polymers Tantalizing promise, largely unfulfilled, haunts gene therapy for human disease. Among several barriers to success, efficient gene delivery to targeted cells remains daunting. Zhou et al. broke one barrier through the synthesis of a new class of delivery vector. The pathway results in the synthesis of highly branched poly(β-amino ester) (HPAE), using a one-pot Michael addition. The transfection efficiency of HPAE over existing vectors improved by as much as three orders of magnitude. Gene delivery by HPAE was able to correct a genetic defect in a recessive dystrophic epidermolysis bullosa graft mouse model. Sci. Adv. 10.1126.sciadv.1600102 (2016).

Adipose‑derived mesenchymal stem cell‑facilitated TRAIL expression in melanoma treatment invitro.

Adipose-derived stem cells (ADSCs) may be useful as an efficient vehicle in cell-based gene therapy of human diseases due to their ability to migrate to disease lesions. This study investigated the ability of ADSC-harbored human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cDNA to facilitate TRAIL expression and induce A375 melanoma cell apoptosis as observed using a Transwell co-culture system. A cell migration assay was used to observe ADSC migration ability. In addition, TRAIL protein expression was successfully detected by western blot analysis in ADSCs after stable transfection of TRAIL cDNA. The Transwell co-culture system data showed that TRAIL-ADSCs could induce A375 cell apoptosis in a dose-dependent manner. At the gene level, the killing activity of TRAIL-ADSCs was associated with activation of caspase-4 and caspase-8. Collectively, the data from the current study provides preclinical support of ADSC-facilitated TRAIL expression in the treatment of melanoma. Further investigation is required to evaluate and confirm the in vivo ability of TRAIL-ADSCs in therapy of melanoma in animal models.

The application of CRISPR/Cas9 technology in the treatment of childhood-onset monogenetic disease

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR/CRISPR-associated nuclease 9), a kind of acquired immune system peculiar to bacteria, has been improved as a new genetic engineering tool. It is known as the scissor of the god because of its huge advantage of simple design, convenient operation, low cost and high efficiency. CRISPR/Cas9 technology has already been widely promoted and applied in gene function research, animal modeling, gene therapy, and other fields. This paper highlights the origin, development and mechanism of CRISPR/Cas9 genome editing technology, and summarizes the latest application in gene therapy for human diseases, especially for the childhood-onset monogenetic diseases, so as to provide a reference for its wider research.(Chin J Lab Med, 2016, 39: 243-245) Key words: CRISPR/Cas9; Gene editing; Monogenetic disease

The molecular mechanism of CRISPR/Cas9 system and its application in gene therapy of human diseases.

CRISPR/Cas system is an adaptive immune system that confers resistance to exogenous virus or plasmid in bacteria and archaea. In recent years, the booming CRISPR/Cas9 genome editing technology modified from type2 CRISPR/Cas adaptive immune system has been widely applied to various research fields of life science and led to revolutionary changes. In this review, we summarize the origin and development of CRISPR/Cas9 genome editing technology as well as its applications in life science research. We focus on the latest application of this system in gene therapy of human diseases and the associated side/off-target effects, which may provide references for researchers in related areas.

Engineered hair follicle mesenchymal stem cells overexpressing controlled-release insulin reverse hyperglycemia in mice with type L diabetes.

Genetically engineered stem cells that overexpress genes encoding therapeutic products can be exploited to correct metabolic disorders by repairing and regenerating diseased organs or restoring their function. Hair follicles are readily accessible and serve as a rich source of autologous stem cells for cell-based gene therapy. Here we isolated mesenchymal stem cells from human hair follicles (HF-MSCs) and engineered them to overexpress the human insulin gene and release human insulin in a time- and dose-dependent manner in response to rapamycin. The engineered HF-MSCs retained their characteristic cell surface markers and retained their potential to differentiate into adipocytes and osteoblasts. When mice with streptozotocin-induced type 1 diabetes were engrafted with these engineered HF-MSCs, these cells expressed and released a dose of human insulin, dramatically reversed hyperglycemia, and significantly reduced death rate. Moreover, the engineered HF-MSCs did not form detectable tumors throughout the 120-day animal tests in our experiment. Our results show that HF-MSCs can be used to safely and efficiently express therapeutic transgenes and therefore show promise for cell-based gene therapy of human disease.

Vector and helper genome rearrangements occur during production of helper-dependent adenoviral vectors.

Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 α promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.

Gene transfer to skeletal muscle by site-specific delivery of electroporation and ultrasound

Transfecting foreign genes into target cells require certain vectors and specific techniques. With more new genes and gene targets for gene therapy are continually being discovered, which provide useful clues for the study of gene function and gene therapy for human disease. However, there still remain a number of important unresolved problems associated with the use of viral and non viral vectors or techniques, such as secondary toxicity, immune response, or low gene transfer efficiency. Therefore, efficient and safe approaches of gene delivery in vivo still need to be found for medical applications. Electroporation or ultrasound (US), which involving electrical pulses or a US field to increase cell membrane permeability, has shown to be an efficient, safe and simple non viral physical method of DNA delivery in vivo and alternative technique in the field of gene therapy. However, the high field strength or energy often required for electroporation or US can result in tissue damage, thus limit their widely clinical applications. In recent years, site-specific gene delivery by electroporation or US has aroused much attention, because of optimized protocols and novel devices using a lower field strength than conventional methods, which has shown high transfection efficiency with minimal tissue damage. In this paper, we reviewed the advancement in the field of electroporation and US for gene delivery, particularly by site-specific delivery into skeletal muscle for gene therapy and their applications in Alzheimer’s disease (AD)’s treatment.

Mesenchymal Stem Cells Transfer Mitochondria to the Cells with Virtually No Mitochondrial Function but Not with Pathogenic mtDNA Mutations

It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine− and bromodeoxyuridine [BrdU]+) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

A syringe‐focused ultrasound device for simultaneous injection of DNA and gene transfer

Although known for its success in ameliorating muscle disorders, ultrasound (US)-mediated gene transfer in skeletal muscle has recently shown potential for treating a wide range of therapeutic applications. The disadvantages of the technique, however, including its relatively low transfer efficiency and toxic effects resulting in tissue damage, have impeded clinical progress. We describe a novel syringe-focused US device of our design that combines simultaneous DNA injection and US irradiation to resolve these issues, achieving improved DNA uptake for high expression and lowering the incidence of tissue injury. The novel, syringe-focused US device was used to introduce purified plasmid DNA encoding human neprilysin (hNEP) into mouse hindlimb skeletal muscle concurrent with US protocol (1.7 MHz, 1 W/cm(2), 1-min exposure time). hNEP expression was measured in local skeletal muscle, blood serum and brain by immunoassay at different post-injection time points. The incidence of muscle fiber damage was subsequently assessed by Evans Blue dye (EBD) uptake using fluorescence microscopy. The syringe-US device at the first time enabled simultaneous combination of DNA transfer and US stimulation gives rise to two months of gene expression of the encoded polypeptide as it is measured, at the same time as withstanding minor tissue damage. In addition, DNA injection without US treatment failed to induce hNEP expression. This new technique provides a simple, safe and efficient nonviral gene delivery approach in skeletal muscle and shows promising applications for gene therapy of human disease, such as Alzheimer's disease.

569. RNAi-Mediated Gene Therapy of Human Diseases

569. RNAi-Mediated Gene Therapy of Human Diseases

Applications of Sleeping Beauty transposons for nonviral gene therapy

Virus-based gene therapy has advanced to clinical trials; however, this approach may result in serious adverse events including oncogenesis and the possibility of triggering fatal immune responses. Nonviral gene delivery approaches have a better safety profile, but their in vivo application has been largely limited in the past due to their inefficient delivery into cells and lack of stable chromosomal integration that is necessary for long-term therapeutic benefit. However, recent advances suggest that the use of Sleeping Beauty transposons, a novel integrating nonviral vector system, are capable of achieving long-lasting therapeutic levels of transgene expression in preclinical settings. These observations and other ongoing relevant studies may unlock the therapeutic potential of nonviral gene therapy for human diseases.

Adeno-Associated Virus-Mediated Gene Transfer in Hematopoietic Stem/Progenitor Cells as a Therapeutic Tool

Hematopoietic stem cells (HSCs) have unique properties of self-renewal, differentiation and proliferation. HSCs are easily accessible, and can be readily delivered back to patients by autologous transplantation, which renders them as attractive targets for ex vivo gene therapy. The adeno-associated virus (AAV) vectors have to date not been associated with any malignant disease, and have gained attention as a potentially safer alternative to the more commonly used retroviral vectors for HSC gene therapy. Although conflicting data exist with regard to HSC transduction by AAV vectors, in this review, we provide an overview of AAV-mediated HSC gene transfer - obstacles as well strategies to improve the transduction efficiency - and the potential use of AAV vectors for gene therapy of human diseases involving HSCs.

380. Studies on the Mechanism of Gene Repair Using Saccharomyces cerevisae as a Model System

Oligonucleotides are being used to direct base changes within genes and can be used as a gene therapy for human disease. While the entire mechanism of the targeted nucleotide exchange (TNE) reaction has yet to be elucidated, there is evidence that it may be separated into at least two phases: the pairing phase, which consists of the MSSO pairing to the target site within the gene, and the repair phase, in which the MSSO directs the repair of the targeted base. This repair phase could possibly be separated into two phases: the first repair event, which is the correction of the strand to which the oligonucleotide is complementary, and the second repair event, in which the corrected strand serves as a template to repair the non-targeted strand. While the proteins that are involved in the pairing phase of the reaction are known, it remains unclear as to which proteins are playing a role in the repair phase of the reaction. Yeast strains deficient in Rad1p and MMR proteins, Msh2p and Msh6p, were used to determine the ability of a modified single-stranded oligonucleotide to direct a base change within a mutant Hygromycin eGFP fusion gene. The data indicate that in the absence of these proteins, the oligonucleotide is incapable of directing the base change, as the targeting frequency is reduced 30 fold and shows very little targeting activity. Upon complementation of Rad1p, using the 2um based plasmid pNF1000, the ability to direct the base change is restored. These data indicate that proteins involved in MMR and NER are playing a role in the TNE reaction and future studies aim to elucidate this role.