Protection from cisplatin-induced hearing loss with lentiviral vector-mediated ectopic expression of the anti-apoptotic protein BCL-XL.

Cisplatin is a highly effective chemotherapeutic agent, but it can cause sensorineural hearing loss (SNHL) in patients. Cisplatin-induced ototoxicity is closely related to the accumulation of reactive oxygen species (ROS) and subsequent death of hair cells (HCs) and spiral ganglion neurons (SGNs). Despite various strategies to combat ototoxicity, only one therapeutic agent has thus far been clinically approved. Therefore, we have developed a gene therapy concept to protect cochlear cells from cisplatin-induced toxicity. Self-inactivating lentiviral (LV) vectors were used to ectopically express various antioxidant enzymes or anti-apoptotic proteins to enhance the cellular ROS scavenging or prevent apoptosis in affected cell types. In direct comparison, anti-apoptotic proteins mediated a stronger reduction in cytotoxicity than antioxidant enzymes. Importantly, overexpression of the most promising candidate, Bcl-xl, achieved an up to 2.5-fold reduction in cisplatin-induced cytotoxicity in HEI-OC1 cells, phoenix auditory neurons, and primary SGN cultures. BCL-XL protected against cisplatin-mediated tissue destruction in cochlear explants. Strikingly, in vivo application of the LV BCL-XL vector improved hearing and increased HC survival in cisplatin-treated mice. In conclusion, we have established a preclinical gene therapy approach to protect mice from cisplatin-induced ototoxicity that has the potential to be translated to clinical use in cancer patients.

Third-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B.

Usher syndrome 1B (USH1B) is a devastating genetic disorder with congenital deafness, loss of balance, and blindness caused by mutations in the myosin-VIIa (MYO7A) gene, for which there is currently no cure. We developed a gene therapy approach addressing the vestibulo-cochlear deficits of USH1B using a third-generation, high-capacity lentiviral vector system capable of delivering the large 6,645-bp MYO7A cDNA. Lentivirally delivered MYO7A and co-encoded dTomato were successfully expressed in the cochlear cell line HEI-OC1. In normal-hearing mice, both cochlea and the vestibular organ were efficiently transduced, and ectopic MYO7A overexpression did not show any adverse effects. In Shaker-1 mice, an USH1B disease model based on Myo7a mutation, cochlear and vestibular hair cells, the main inner ear cell types affected in USH1B, were successfully transduced. In homozygous mutant mice, delivery of MYO7A at postnatal day 16 resulted in a trend for partial recovery of auditory function and in strongly reduced balance deficits. Heterozygous mutant mice were found to develop severe hearing loss at 6 months of age without balance deficits, and lentiviral MYO7A gene therapy completely rescued hearing to wild-type hearing thresholds. In summary, this study demonstrates improved hearing and balance function through lentiviral gene therapy in the inner ear.

Efficient Genetic Safety Switches for Future Application of iPSC-Derived Cell Transplants.

Induced pluripotent stem cell (iPSC)-derived cell products hold great promise as a potential cell source in personalized medicine. As concerns about the potential risk of graft-related severe adverse events, such as tumor formation from residual pluripotent cells, currently restrict their applicability, we established an optimized tool for therapeutic intervention that allows drug-controlled, specific and selective ablation of either iPSCs or the whole graft through genetic safety switches. To identify the best working system, different tools for genetic iPSC modification, promoters to express safety switches and different safety switches were combined. Suicide effects were slightly stronger when the suicide gene was delivered through lentiviral (LV) vectors compared to integration into the AAVS1 locus through TALEN technology. An optimized HSV-thymidine kinase and the inducible Caspase 9 both mediated drug-induced, efficient in vitro elimination of transgene-positive iPSCs. Choice of promoter allowed selective elimination of distinct populations within the graft: the hOct4 short response element restricted transgene expression to iPSCs, while the CAGs promoter ubiquitously drove expression in iPSCs and their progeny. Remarkably, both safety switches were able to prevent in vivo teratoma development and even effectively eliminated established teratomas formed by LV CAGs-transgenic iPSCs. These optimized tools to increase safety provide an important step towards clinical application of iPSC-derived transplants.

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.

Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes.

Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47phox subunit of the oxidase complex carry the deletion c.75_76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47phox) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach.

Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application.

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy.

Uncoupling the Oncogenic Engine.

Inhibition of oncogenic signaling and correction of aberrant metabolic processes may be key paradigms to eliminate cancer cells. The high incidence of activating RAS mutations and hyperactivated ERK1/2 signaling observed in many human tumors and the lack of effective targeted therapies to elicit long-term inhibition of the RAS-ERK1/2 signaling pathway add to the importance of discovering novel strategies to treat malignancies characterized by elevated RAS-ERK1/2 signaling. In this review, we describe connections between oncogenic signaling and cancer cell metabolism and how these links may be exploited for novel modern molecular medicine approaches. Cancer Res; 77(22); 6060-4. ©2017 AACR.

Correction to: Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

The authors wish to apologize for an error within the scale bar of the microarray heatmap in Additional File 5 of the supplementary information. Two values were incorrectly displayed on the scale bar (11 instead of 10 and 13 instead of 12).

Integrating Vectors for Gene Therapy and Clonal Tracking of Engineered Hematopoiesis.

Gene therapy using autologous or allogeneic cells offers promising possibilities to treat inherited and acquired diseases, ideally leading to a long-lasting therapeutic correction. This article summarizes efforts that use integrating vectors derived from retroviruses and transposons, and briefly explains integrating vector biology and integration site analysis and recent successful application of this technology in clinical trials. Moreover, outlined is how these vectors can be used for cancer gene discovery and clonal tracking of benign and malignant hematopoiesis to gain insights into the dynamics of hematopoiesis.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

BACKGROUND: Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. RESULTS: In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. CONCLUSIONS: We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Viral and Synthetic RNA Vector Technologies and Applications.

Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy.

Retrovirus-based vectors for transient and permanent cell modification.

Retroviral vectors are commonly employed for long-term transgene expression via integrating vector technology. However, three alternative retrovirus-based platforms are currently available that allow transient cell modification. Gene expression can be mediated from either episomal DNA or RNA templates, or selected proteins can be directly transferred through retroviral nanoparticles. The different technologies are functionally graded with respect to safety, expression magnitude and expression duration. Improvement of the initial technologies, including modification of vector designs, targeted increase in expression strength and duration as well as improved safety characteristics, has allowed maturation of retroviral systems into efficient and promising tools that meet the technological demands of a wide variety of potential application areas.

Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while avoiding integration, the developed technology is of great significance for a broad panel of applications, including transcription-factor-based induced cell fate conversion and controlled transfer of genetically encoded RNA- or protein-based drugs.

Viral and non-viral approaches for transient delivery of mRNA and proteins.

The transient delivery of gene products (RNA or proteins) is not a biotechnological invention but rather an evolutionarily conserved process underlying and regulating a variety of biological functions. On the basis of insights into the underlying mechanisms, several viral and cell-based approaches have been developed for the delivery of RNA or proteins. Prominent applications include the induction of major biological or therapeutic effects on the basis of "hit-and-run" mechanisms, such as vaccination, cell fate modification (reprogramming, differentiation), control of cell trafficking, enhancement of cell regeneration, and genome engineering using sequence-specific recombinases or nucleases. Ideally, procedures for delivery of RNA or proteins should be targeted to specific cells, overcome biophysical hurdles without harming cellular integrity, circumvent the various alarm signals of the innate immune system, allow dose-controlled delivery of functional biomacromolecules, and avoid the induction of an adaptive immune response. Here we review the current state of approaches for the delivery of mRNA and proteins with a focus on RNA viruses, virus-like particles including retrovirus particle-mediated transfer of mRNA or proteins, extracellular vesicles, and cell-penetrating peptides. The basic concepts and recent advances are put into perspective in the context of potential limitations of the technologies and strategies to overcome cellular barriers and defense mechanisms.