Gene Therapy Of Genetic Diseases Research Articles

Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice

BackgroundRecent advances in gene editing technology have opened up new avenues for in vivo gene therapy, which holds great promise as a potential treatment method for dilated cardiomyopathy (DCM). The CRISPR-Cas13 system has been shown to be an effective tool for knocking down RNA expression in mammalian cells. PspCas13b, a type VI-B effector that can be packed into adeno-associated viruses and improve RNA knockdown efficiency, is a potential treatment for diseases characterized by abnormal gene expression.ResultsUsing PspCas13b, we were able to efficiently and specifically knockdown the mutant transcripts in the AC16 cell line carrying the heterozygous human TNNT2R141W (hTNNT2R141W) mutation. We used adeno-associated virus vector serotype 9 to deliver PspCas13b with specific single guide RNA into the hTNNT2R141W transgenic DCM mouse model, effectively knocking down hTNNT2R141W transcript expression. PspCas13b-mediated knockdown significantly increased myofilament sensitivity to Ca2+, improved cardiac function, and reduced myocardial fibrosis in hTNNT2R141W DCM mice.ConclusionsThese findings suggest that targeting genes through Cas13b is a promising approach for in vivo gene therapy for genetic diseases caused by aberrant gene expression. Our study provides further evidence of Cas13b’s application in genetic disease therapy and paves the way for future applicability of genetic therapies for cardiomyopathy.

Recent advances in hematopoietic gene therapy for genetic disorders

Hematopoietic gene therapy is based on the transplantation of gene-modified autologous hematopoietic stem cells and since the inception of this approach, many technological and medical improvements have been achieved. This review focuses on the clinical studies that have used hematopoietic gene therapy to successfully treat several rare and severe genetic disorders of the blood or immune system as well as some non-hematological diseases. Today, in some cases hematopoietic gene therapy has progressed to the point of being equal to, or better than, allogeneic bone marrow transplant. In others, further improvements are needed to obtain more consistent efficacy or to reduce the risks posed by vectors or protocols. Several hematopoietic gene therapy products showing both long-term efficacy and safety have reached the market, but economic considerations challenge the possibility of patient access to novel disease-modifying therapies. © 2023 Published by Elsevier Masson SAS on behalf of French Society of Pediatrics.

Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases.

The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases. However, the susceptibility of nucleic acid drugs to degradation by nucleic acid endonucleases necessitates the development of functional vectors capable of protecting the nucleic acids from enzymatic degradation while ensuring safety and effectiveness. This review explores the biomedical potential of non-viral vector-based CRISPR/Cas9 systems for treating genetic diseases. Furthermore, it provides a comprehensive overview of recent advances in viral and non-viral vector-based gene therapy for genetic disorders, including preclinical and clinical study insights. Additionally, the review analyzes the current limitations of these delivery systems and proposes avenues for developing novel nano-delivery platforms.

Overview of Retinal Gene Therapy: Current Status and Future Challenges.

The success of the first Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved gene therapy for genetic disease, voretigene neparovovec-rzyl, (Luxturna) has helped pave the way for development of retinal gene therapies to target other genetic and acquired forms of blindness. Gene therapy trials are now taking place in multiple continents and numerous countries, they use several different gene transfer reagents ("vectors"), studies have used several different routes of administration, and different strategies are being tested in interventional studies with promising results. The future has never been brighter for individuals with retinal degeneration. Here and in the literature cited below, we summarize the state-of-the-art of retinal gene therapy and consider some of the questions and challenges that lie ahead.

Ribosome biogenesis and ribosome therapy in cancer cells

Introduction: The process of protein synthesis is a vital process for all kingdoms of life. The ribosome is a ribonu­cleoprotein complex that reads the genetic code, from messenger RNA (mRNA) to produce proteins and to tightly regulate and ensure cells growth. The fact that numerous diseases are caused by defect during the ribosome biogenesis is important to understand this pathway.
 Materials and methods: We have analyzed the literature for ribosome biogenesis and its links with different diseases which have been found.
 Results and discussion: We have discussed the key aspect of human ribosome biogenesis and its links to diseases. We have also proposed the potential of applying this knowledge to the development of a ribosomal stress-based cancer therapy.
 Conclusion: Major challenges in the future will be to determine factors which play a pivotal role during ribosome bio­genesis. Therefore, more anti-cancer drugs and gene therapy for genetic diseases will be developed against ribosomal biogenesis in the coming years.
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Resveratrol Delivery via Gene Therapy: Entering the Modern Era

Resveratrol is a natural compound (an antioxidant) and exhibits numerous therapeutic activities. From a pharmacokinetic perspective, it is unclear whether resveratrol targets the site of action after oral administration because of quick metabolism and excretion that creates doubt on the biological application of the high doses characteristically used for clinical trials. However, these limitations act as a barrier and a challenge for the development of new delivery systems. Recently, gene delivery offers various advantages and has provided treatment options for diseases that are beyond the reach of traditional approaches. The objective of gene therapy for genetic diseases is to achieve durable expression of the therapeutic gene at a level sufficient to alleviate or cure disease symptoms with minimal adverse events. The perception of the molecular and cellular mechanisms steering to therapy and vector-related hindrances have caused in the progress of extremely complex gene delivery with enhanced protection and effectiveness. With the help of gene therapy, it could be possible to target the delivery of resveratrol directly into the host cells and bypass its pharmacokinetic limitations and enhancement of its therapeutic effect. This review is to provide a holistic view of the development of resveratrol gene treatment as a powerful option to treat various deadly diseases.

Optimization of Synthesis of the Amino Lipid ECO for Effective Delivery of Nucleic Acids.

Nucleic acids are promising for a variety of therapies, such as cancer therapy and the gene therapy of genetic disorders. The therapeutic efficacy of nucleic acids is reliant on the ability of their efficient delivery to the cytosol of the target cells. Amino lipids have been developed to aid in the cytosolic delivery of nucleic acids. This work reports a new and efficient synthetic pathway for the lipid carrier, (1-aminoethyl) iminobis [N-(oleicylcysteinyl-1-amino-ethyl)propionamide] (ECO). The previous synthesis of the ECO was inefficient and presented poor product quality control. A solution-phase synthesis of the ECO was explored, and each intermediate product was characterized with better quality control. The ECO was synthesized with a relatively high yield and high purity. The formulations of the ECO nanoparticles were made with siRNA, miRNA, or plasmid DNA, and characterized. The transfection efficiency of the nanoparticles was evaluated in vitro over a range of N/P ratios. The nanoparticles were consistent in size with previous formulations and had primarily a positive zeta potential. The ECO/siLuc nanoparticles resulted in potent luciferase silencing with minimal cytotoxicity. The ECO/miR-200c nanoparticles mediated the efficient delivery of miR-200c into the target cells. The ECO/pCMV-GFP nanoparticles resulted in substantial GFP expression upon transfection. These results demonstrate that the solution-phase synthetic pathway produced pure ECO for the efficient intracellular delivery of nucleic acids without size limitation.

Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases.

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

ACBE, a new base editor for simultaneous C-to-T and A-to-G substitutions in mammalian systems

BackgroundMany favorable traits of crops and livestock and human genetic diseases arise from multiple single nucleotide polymorphisms or multiple point mutations with heterogeneous base substitutions at the same locus. Current cytosine or adenine base editors can only accomplish C-to-T (G-to-A) or A-to-G (T-to-C) substitutions in the windows of target genomic sites of organisms; therefore, there is a need to develop base editors that can simultaneously achieve C-to-T and A-to-G substitutions at the targeting site.ResultsIn this study, a novel fusion adenine and cytosine base editor (ACBE) was generated by fusing a heterodimer of TadA (ecTadAWT/*) and an activation-induced cytidine deaminase (AID) to the N- and C-terminals of Cas9 nickase (nCas9), respectively. ACBE could simultaneously induce C-to-T and A-to-G base editing at the same target site, which were verified in HEK293-EGFP reporter cell line and 45 endogenous gene loci of HEK293 cells. Moreover, the ACBE could accomplish simultaneous point mutations of C-to-T and A-to-G in primary somatic cells (mouse embryonic fibroblasts and porcine fetal fibroblasts) in an applicable efficiency. Furthermore, the spacer length of sgRNA and the length of linker could influence the dual base editing activity, which provided a direction to optimize the ACBE system.ConclusionThe newly developed ACBE would expand base editor toolkits and should promote the generation of animals and the gene therapy of genetic diseases with heterogeneous point mutations.

Gene Therapy Options as New Treatment for Inherited Peripheral Neuropathy.

Inherited peripheral neuropathy (IPN) is caused by heterogeneous genetic mutations in more than 100 genes. So far, several treatment options for IPN have been developed and clinically evaluated using small molecules. However, gene therapy-based therapeutic strategies have not been aggressively investigated, likely due to the complexities of inheritance in IPN. Indeed, because the majority of the causative mutations of IPN lead to gain-of-function rather than loss-of-function, developing a therapeutic strategy is more difficult, especially considering gene therapy for genetic diseases began with the simple idea of replacing a defective gene with a functional copy. Recent advances in gene manipulation technology have brought novel approaches to gene therapy and its clinical application for IPN treatment. For example, in addition to the classically used gene replacement for mutant genes in recessively inherited IPN, other techniques including gene addition to modify the disease phenotype, modulations of target gene expression, and techniques to edit mutant genes have been developed and evaluated as potent therapeutic strategies for dominantly inherited IPN. In this review, the current status of gene therapy for IPN and future perspectives will be discussed.

Poly(1-vinylimidazole) Prospects in Gene Delivery

Polymeric amines are being studied intensively as components of systems for gene delivery in genetic engineering and gene therapy of genetic disorders, including cancer. Despite remarkable achievements in the field, polymeric amines, such as polyethyleneimine, show some disadvantages. Strong interaction between the amine-containing polymer and nucleic acid hampers the release of nucleic acid in the cell cytoplasm. Amine groups can interact with the cell membrane which results in cell death. These limitations of polymeric amines stimulated an investigation of new structures for gene delivery. Imidazole-containing polymers have attracted attention as lesser basic substances, while they are able to interact with polymeric acids. Further development of imidazole-based gene delivery agents requires knowledge about some fundamental aspects of interaction between nucleic acids, and polymeric imidazoles. In this work, we studied the complexation of poly(1-vinylimidazole) and oligomeric DNA. We found that the number of active sites capable of binding with negatively charged phosphate groups is comparable with the number of protonated imidazole units in the case of high molecular weight polymer. The increase in polymer charge by 1-bromopropane quaternizating 1%−5% imidazole units or by decreasing the pH to 6.5−7 considerably increased the ability of poly(1-vinylimidazole) to interact with oligonucleotides. The pH sensitivity of this interaction is interesting for cancer gene therapy because the tumours have a lowered intercellular pH (stable oligonucleotide complex) and a higher extracellular pH which can lead to complex dissociation. Minimal critical length for complexation of quaternized poly(1-vinylimidazole) and DNA is below eight units which corresponds to polymers with amine groups. Fluorescence-tagged poly(1-vinylimidazole) samples were obtained and their potential for monitoring the polymer and polymer-oligonucleotide complex internalization into living cells was demonstrated.

First gene therapy for genetic disease approved

A shot of DNA-filled viruses injected directly into the eye is now the first gene therapy approved by the U.S. Food & Drug Administration to treat a genetic disease. The therapy, called Luxturna, was developed by Philadelphia-based Spark Therapeutics and treats a form of inherited blindness. “I believe gene therapy will become a mainstay in treating, and maybe curing, many of our most devastating and intractable illnesses,” FDA Commissioner Scott Gottlieb said. “We’re at a turning point when it comes to this novel form of therapy.” During a press briefing, Gottlieb heralded Luxturna as FDA’s third gene therapy approval in 2017. The first two were CAR T-cell immunotherapies, in which a patient’s immune cells are removed, injected with DNA outside the body, and returned to the bloodstream to fight cancer cells. Luxturna, though, is the first treatment that fits the traditional notion of gene therapy, whereby DNA-filled viruses are injected

CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice.

Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.

MRNA trans-splicing in gene therapy for genetic diseases.

Spliceosome‐mediated RNA trans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post‐transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre‐mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487–498. doi: 10.1002/wrna.1347For further resources related to this article, please visit the WIREs website.

Genetic Correction of Induced Pluripotent Stem Cells From a Deaf Patient With MYO7A Mutation Results in Morphologic and Functional Recovery of the Derived Hair Cell-Like Cells.

The genetic correction of induced pluripotent stem cells (iPSCs) induced from somatic cells of patients with sensorineural hearing loss (caused by hereditary factors) is a promising method for its treatment. The correction of gene mutations in iPSCs could restore the normal function of cells and provide a rich source of cells for transplantation. In the present study, iPSCs were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T; P-iPSCs), the asymptomatic father of the patient (MYO7A c.1184G>A mutation; CF-iPSCs), and a normal donor (MYO7A(WT/WT); C-iPSCs). One of MYO7A mutation sites (c.4118C>T) in the P-iPSCs was corrected using CRISPR/Cas9. The corrected iPSCs (CP-iPSCs) retained cell pluripotency and normal karyotypes. Hair cell-like cells induced from CP-iPSCs showed restored organization of stereocilia-like protrusions; moreover, the electrophysiological function of these cells was similar to that of cells induced from C-iPSCs and CF-iPSCs. These results might facilitate the development of iPSC-based gene therapy for genetic disorders. Induced pluripotent stem cells (iPSCs) were generated from a deaf patient with compound heterozygous MYO7A mutations (c.1184G>A and c.4118C>T). One of the MYO7A mutation sites (c.4118C>T) in the iPSCs was corrected using CRISPR/Cas9. The genetic correction of MYO7A mutation resulted in morphologic and functional recovery of hair cell-like cells derived from iPSCs. These findings confirm the hypothesis that MYO7A plays an important role in the assembly of stereocilia into stereociliary bundles. Thus, the present study might provide further insight into the pathogenesis of sensorineural hearing loss and facilitate the development of therapeutic strategies against monogenic disease through the genetic repair of patient-specific iPSCs.

Progress in gene therapy for genetic diseases

Progress in gene therapy for genetic diseases

290. Baboon Envelope Pseudo Typed Lentiviral Vectors Mediate High-Level Gene Transfer in Human B Cells Allowing Secretion of FIX at Therapeutic Levels in NSG Mouse

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction. In addition, long-lasting transgene expression in B cells is of particular interest for immunotherapy by its potential to induce specific immune activation or tolerance. Moreover, B cells are potent specialized protein secreting cells. One obstacle though is that primary B cells are poorly transduced by VSVG pseudotyped lentiviral vectors (LVs) even when stimulated through the BCR to induce proliferation. We demonstrated that this is due to the poor expression of the low density lipid (LDL) receptor, identified as the VSV receptor (Amirache et al, 2014).Since we recently showed that baboon retroviral envelope pseudotyped LVs (BaEV) outperform VSV-G-LVs for gene transfer into cytokine-stimulated and resting hematopoietic stem cells, we evaluated them here for gene transfer into primary human B cells. Upon B cell receptor stimulation, BaEV-LVs transduced up to 70% of the B cells. As expected, VSV-G-LVs were unable to transduce activated nor unstimulated resting human B cells efficiently, even at high vector doses (MOI= 100, VSV-G-LV < 5% transduction). Remarkably, BaEV-LVs permitted highly efficient transduction of 30% of resting B cells. Moreover, they transduced as well memory as naive B cells without inducing phenotypic changes. In addition, BaEV-LVs permitted up to 80% transduction of human plasmocytes. Adaptive transfer of mature BaEV-LV transduced human B cells into NSG mice allowed differentiation into plasmablasts and plasma B cells, both characterized by a high level gene marking in the lymphnodes, spleen and bone marrow. These results encouraged us to evaluate BaEV-LV mediated gene transfer of Factor IX into human B cells for treatment of heamophelia, a severe blood disorder. We produced high-titer BaEV-LVs carrying a codon optimized FIX with a hyperactivating FIX-R338L mutation. These BaEV-FIX-LVs efficiently transduced plasmocytic B cell lines leading to high-level FIX secretion (20-70% of normal levels in human serum). Transduction of primary human B cells followed by adaptive transfer into NSG mice resulted in therapeutic levels of FIX in the serum (7-25% of normal FIX levels) at 3 to 4 weeks of engraftment. Moreover, a faster coagulation of the blood proved that FIX was functional.Concluding, the BaEV-LVs might represent in the future valuable tools for therapeutic protein secretion from autologous B cells for treatment of heamophelia and other disorders in vivo.

Perspectives on stem cell gene therapy for genetic disorders

Gene transfer into haematopoietic stem cells (HSCs) has now come to the stage where it can be recognized as a form of established therapy for patients with a certain range of genetic diseases. Primary immunodeficiency disease (PID) represents an ideal pathological target for this type of genetic medicine, that is HSC gene therapy, as exemplified well by the successful cases with adenosine‐deaminase (ADA) deficiency. Other diseases including X‐linked severe combined immunodeficiency disease (X‐SCID), chronic granulomatous disease (CGD) and Wiskott–Aldrich syndrome (WAS) have also been the targets of gene therapy, but the efficacy reportedly varies considerably. In addition, all those diseases except ADA deficiency have been demonstrated to be subject to an inherent risk of insertional leukaemogenesis as long as gene transfer accompanies genomic insertion. Researchers therefore should continue to aim for further improvement of HSC gene therapy to maximize both safety and efficacy. To achieve this aim, an ideal model system is desired, by which faithful recapitulation of the disease‐related pathophysiology is feasible. Recently, a new model system possibly constituting such an ideal platform has come to reality; that is, patient‐/disease‐specific induced pluripotent stem cells (iPSCs). iPSCs should particularly be useful for modelling genetic disorders, because of their excellent potential to differentiate into any types of somatic cells while retaining the patient‐specific genetic mutations. These model cells should allow testing various kinds of genetic manipulation and also detailed analysis of the target cells before/after differentiation, thus providing a disease‐specific platform for preclinical studies to test safety and efficacy of each gene‐modification procedure.

Genomic discovery of potent chromatin insulators for human gene therapy.

Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

Precise Correction of the Dystrophin Gene in Duchenne Muscular Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and CRISPR-Cas9

SummaryDuchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.