Gene Therapy Of Monogenic Diseases Research Articles

Translational potential of base-editing tools for gene therapy of monogenic diseases.

Millions of people worldwide have rare genetic diseases that are caused by various mutations in DNA sequence. Classic treatments of rare genetic diseases are often ineffective, and therefore great hopes are placed on gene-editing methods. A DNA base–editing system based on nCas9 (Cas9 with a nickase activity) or dCas9 (a catalytically inactive DNA-targeting Cas9 enzyme) enables editing without double-strand breaks. These tools are constantly being improved, which increases their potential usefulness for therapies. In this review, we describe the main types of base-editing systems and their application to the treatment of monogenic diseases in experiments in vitro and in vivo. Additionally, to understand the therapeutic potential of these systems, the advantages and disadvantages of base-editing systems are examined.

Cas-Based Systems for RNA Editing in Gene Therapy of Monogenic Diseases: In Vitro and in Vivo Application and Translational Potential.

Rare genetic diseases reduce quality of life and can significantly shorten the lifespan. There are few effective treatment options for these diseases, and existing therapeutic strategies often represent only supportive or palliative care. Therefore, designing genetic-engineering technologies for the treatment of genetic diseases is urgently needed. Rapid advances in genetic editing technologies based on programmable nucleases and in the engineering of gene delivery systems have made it possible to conduct several dozen successful clinical trials; however, the risk of numerous side effects caused by off-target double-strand breaks limits the use of these technologies in the clinic. Development of adenine-to-inosine (A-to-I) and cytosine-to-uracil (C-to-U) RNA-editing systems based on dCas13 enables editing at the transcriptional level without double-strand breaks in DNA. In this review, we discuss recent progress in the application of these technologies in in vitro and in vivo experiments. The main strategies for improving RNA-editing tools by increasing their efficiency and specificity are described as well. These data allow us to outline the prospects of base-editing systems for clinical application.

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.

Reprogramming Human T Cell Function and Specificity With Non–Viral Genome Targeting

TL Roth, C Puig–Saus, R Yu. Nature. 2018;559(7714):405–409 To develop and optimize nonviral CRISPR-Cas9 genome editing for primary T cells and apply this technique in part to correct a pathogenic mutation in a monogenic autoimmune disease. Using CRISPR-Cas9, primary human T cells from healthy donors and a family with monogenic autoimmune disease were engineered ex vivo along with other important proofs of concept. The CRISPR-Cas9 system was first described as a prokaryotic adaptive immune response, and it has since been widely used as a gene editing tool. CRISPR sequences identify homologous areas in the DNA, and Cas9 creates precise double-stranded breaks. Double-stranded breaks are repaired by either nonhomologous end joining or homology-directed repair (HDR). HDR is less error prone, and by using CRISPR-Cas9 with HDR templates, transgenes can be integrated into the targeted genome with precision. In this study, an electroporation method was optimized for ex vivo primary human T cells to allow for uptake of CRISPR-Cas9 ribonucleoprotein complexes and large (>1 kb) double-stranded DNA (dsDNA) templates for HDR. The technique allowed for optimized cell viability along with efficiency under …

291. CD26 Inhibition Enhances Chimerism of Lentivirally Transduced Hematopoietic Stem Cells in a Non-Myeloablative Setting

Despite the progress of gene therapy for monogenic diseases over the last years, the engrafting capacity of gene-modified cells, as well as constant and high-level gene expression in vivo, require further optimization, especially when a reduced intensity conditioning is mostly preferred. Culture conditions applied for effective gene transfer lead to significant loss of long-term repopulating cells and ultimately, impaired engraftment. The ex vivo inhibition of CD26 peptidase activity with Diprotin A was shown to improve homing and engraftment of unmanipulated hematopoietic stem cells (HSCs). We here, sought to assess the homing and engrafting capacity of lentivirally GFP-transduced murine bone marrow (BM) cells under competitive niche settings generated by a non-myeloablative conditioning. Following CD26 inhibition with 5mM Diprotin A of GFP-transduced murine HSCs, we assessed migration towards SDF-1 in transwell systems and the engraftment capacity after partial myeloablation (Busulfan 100mg/kg, equivalent to 8mg/kg in humans) in a syngeneic mouse model, allowing for donor chimera detection (C57Bl6-CD45.2+ donors/PepBoy-CD45.1+ recipients). We also investigated the possible effects of Diprotin A on gene transfer efficiency and transgene expression in bulk and clonogenic cultures of HSCs. In vitro, Diprotin A significantly increased the expression of the CXCR4 receptor (25.6±0.75 vs 16.4±1.02, P=0.02), as well as the %migration of gene-modified HSCs towards SDF-1 over untreated transduced cells (71.78±3.79% vs 55.71±5.34%, P=0.03), implying a potentially enhanced engrafting dynamic in the BM. Indeed, in vivo, the Diprotin A-treated transduced HSCs exhibited faster hematologic reconstitution by, at least, one week (P=0.03) and both superior long-term engraftment and GFP expression in all hematopoietic tissues (peripheral blood, BM, spleen) of the recipients, over non-Diprotin A-treated transduced cells (blood 4th month post-transplant, %CD45.2+: 77.26±5.26 vs 13.11±11.69, P=0.0002; %GFP+: 30.21±6.86 vs 3.9±2.33, P=0.03). The increased GFP expression observed in vivo in the Diprotin A cohort reflected the enhanced engraftment rates, since Diprotin A per se did not affect gene transfer efficiency or transgene expression prior to transplantation (%transduction with Diprotin A: 93±3.05 vs without Diprotin A: 89.9±7.47, P=0.73; %GFP in pools of colonies with Diprotin A: 74.8±7.14 vs without Diprotin A: 72.1±8.45, P=0.81). Upon sacrifice, the Diprotin A animals displayed sustained gene marking with ~1 vector copy in all hematopoietic tissues, as opposed to almost undetectable vector copy number (VCN) in the non-Diprotin A cohort. Likewise, individual colonies from the chimeric BM, demonstrated significantly higher VCN in the Diprotin A mice (2.8±0.48 vs 0.37±0.37, P=0.01). Overall, the ex vivo treatment with Diprotin A seems to abrogate the negative impact of culture conditions, allowing for enhanced donor chimerism under partial myeloablation and consequently, increased gene marking in vivo. This ex vivo, easily applicable approach may serve to overcome major constraints for the clinical implementation of gene therapy should the data be confirmed with human CD34+ cells.

Current progress on gene therapy for primary immunodeficiencies

Primary immunodeficiencies have played a major role in the development of gene therapy for monogenic diseases of the bone marrow. The last decade has seen convincing evidence of long-term disease correction as a result of ex vivo viral vector-mediated gene transfer into autologous haematopoietic stem cells. The success of these early studies has been balanced by the development of vector-related insertional mutagenic events. More recently the use of alternative vector designs with self-inactivating designs, which have an improved safety profile has led to the initiation of a wave of new studies that are showing early signs of efficacy. The ongoing development of safer vector platforms and gene-correction technologies together with improvements in cell-transduction techniques and optimised conditioning regimes is likely to make gene therapy amenable for a greater number of PIDs. If long-term efficacy and safety are shown, gene therapy will become a standard treatment option for specific forms of PID.

Alpharetroviral Vector-mediated Gene Therapy for X-CGD: Functional Correction and Lack of Aberrant Splicing

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.

Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease

AbstractHuman induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Towards Artificial Metallonucleases for Gene Therapy: Recent Advances and New Perspectives

The process of DNA targeting or repair of mutated genes within the cell, induced by specifically positioned double-strand cleavage of DNA near the mutated sequence, can be applied for gene therapy of monogenic diseases. For this purpose, highly specific artificial metallonucleases are developed. They are expected to be important future tools of modern genetics. The present state of art and strategies of research are summarized, including protein engineering and artificial 'chemical' nucleases. From the results, we learn about the basic role of the metal ions and the various ligands, and about the DNA binding and cleavage mechanism. The results collected provide useful guidance for engineering highly controlled enzymes for use in gene therapy.

474. Deletion of All Membranous Genes of Recombinant Sendai Virus Results in the Dramatic Reduction of Toxicity for Pulmonary Gene Transfer in Neonatal Mice

Top of pageAbstract Background and Aim: We previously demonstrated the dramatically high efficiency of recombinant Sendai virus (SeV) for pulmonary gene transfer, 1,000-100,000 fold over that via adenoviruses, and suggested that its possible application for gene therapy of cystic fibrosis (CF) (Yonemitsu Y et al. Nature Biotechnol 2000). As widely known, however, experimental inoculation of SeV exhibits inflammatory and immune responses in rodent lung, frequently resulted in the death of animals. For future directions of pulmonary gene therapy for monogenic diseases, including CF, neonatal gene transfer is ideal, because the lung disease may be in reversible. We recently developed a new and advanced design of SeVs for clinical application, namely F-deficient SeV (SeV/dF), temperature-sensitive SeV/dF (tsSeV/dF), and SeV deleted with all membranous genes (SeV/dFdMdHN). Here we characterized these new constructs for pulmonary gene transfer in neonatal mice, especially referring to toxicity. Method and Results: Recombinant SeVs expressing GFP (2.5 |[times]| 10|[and]|6 pfu in 10|[mu]|l PBS /neonate or 2.5 |[times]| 10|[and]|7 pfu in 100|[mu]|l PBS /adult) was placed into the nasal cavity and the solution was sniffed into the lungs of neonate (day 0) or adult (8 weeks old, female) of ICR (CD-1) mice. The efficiency of gene transfer and the level of gene expression were also monitored using same amount of SeVs expressing luciferase. The peak of gene expression in lung of neonatal mice was at 48 h and decreased gradually, and then almost disappeared at 14 days after infection in all constructs, similar to the finding found in adult mice. Increase of the body weight of neonates transfected with tsSeV/dF or SeV/dFdMdHN, however, was significantly larger than those of animals transfected with wild-SeV or SeV/dF, and the effect was dramatic in use of SeV/dFdMdHN. Mortality rate was dramatically diminished in the mice translated SeV/dFdMdHN (survival rate=100%, n=43) compared with wild-SeV (25%, n=36) or tsSeV/dF (75%, n=44). Assessments of immunological parameters are now underway. Discussion and Conclusion: Our current study revealed that deletion of all membranous genes of recombinant SeV dramatically reduced the toxicity for pulmonary gene transfer in neonatal mice. In conclusion, the potential of pulmonary gene thansfer using SeV/dFdMdHN warrants for further intervention to develop more efficient therapy of neonatal lung diseases including cystic fibrosis.

The Economics of Gene Therapy and of Pharmacogenetics

This paper focuses on the economic issues arising from two uses of genomics: 1) the development of gene therapy; 2) and use of pharmacogenetics to identify a patient's genotype before treatment to exclude those who will not benefit or who may be harmed. We conclude that private-sector investment aimed at developing gene therapy for monogenic diseases is likely to be socially suboptimal. Short-term administration regimens yielding long-term therapeutic benefits are likely to meet payer resistance to large “one-off” costs because of budget constraints or, in competitive systems, concerns that the savings would accrue to future insurers or would attract high-cost patients. For some monogenic diseases, patient numbers may be too small to support commercial development without changes to orphan drug legislation or payer willingness to accept higher cost-effectiveness thresholds. In the case of pharmacogenetics, we conclude that it can often be socially optimal to test before treatment, particularly if the proportion of nonresponders is high, if there is a potential for serious adverse reactions, or if the test is inexpensive. Genetic testing that fragments the patient population could reduce incentives for R&D unless prices are adjusted to reflect the higher expected benefits of targeted treatment per patient. Even in situations where prices are adjusted, patient populations may be too small to make commercial development viable. This problem with small numbers is analogous to that associated with gene therapy for monogenic diseases and may require similar remedies if society values developing treatments for these diseases.