Retroviral Integrations in Gene Therapy Trials

Abstract

γ-Retroviral and lentiviral vectors allow the permanent integration of a therapeutic transgene in target cells and have provided in the last decade a delivery platform for several successful gene therapy (GT) clinical approaches. However, the occurrence of adverse events due to insertional mutagenesis in GT treated patients poses a strong challenge to the scientific community to identify the mechanisms at the basis of vector-driven genotoxicity. Along the last decade, the study of retroviral integration sites became a fundamental tool to monitor vector–host interaction in patients overtime. This review is aimed at critically revising the data derived from insertional profiling, with a particular focus on the evidences collected from GT clinical trials. We discuss the controversies and open issues associated to the interpretation of integration site analysis during patient's follow up, with an update on the latest results derived from the use of high-throughput technologies. Finally, we provide a perspective on the future technical development and on the application of these studies to address broader biological questions, from basic virology to human hematopoiesis.

BET proteins promote efficient murine leukemia virus integration at transcription start sites

“…In clinical trials, the use of γ-retroviral vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred associated with insertion of MLV-based vectors near protooncogenes (reviewed in refs. [15][16][17][18]. The identification of cellular factors for γ-retroviruses may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.…”

Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

“…4,[32][33][34] However, these methods sometimes lack quantitative standards and may not accurately measure clone size in all instances, particularly when restriction enzyme digestion is used to facilitate adapter ligation. 13,17,35 Recently, random DNA shearing by sonication and count of shear site was introduced and appear to have better accuracy and sensitivity. 20,21,23,28,36,37 We further improved this method by minimizing the number of exponential PCR cycles used for template generation and by including an internal control sample with a relatively high number of quantitatively defined VIS.…”

“…Another approach is to use direct genome sequencing without PCR, which may be more feasible as the technology and computing power improves. 35 When the qsLAM PCR assay was used to evaluate human CD34+ cells transduced with a clinical lentiviral vector, there were no clones that comprised more than 2% of the population as defined by the internal control signal strength. This is expected because it is known that many CD34+ cells are not repopulating cells and that the transduced bulk graft is highly polyclonal.…”

Replacing bad (F)actors: hemophilia

“…Although recombinant lentivectors are among the most well-studied gene transfer technologies and are being used in several ongoing clinical trials, there is the theoretical potential for insertional mutagenesis, a topic that has been well reviewed by others. 30 Third, the HSC compartment is mostly quiescent and is not efficiently transduced by ␥-retroviruses. However, unlike ␥-retroviruses, lentivectors efficiently transduce quiescent cell populations.…”

“…The first, as mentioned earlier, is the general understanding of lentivector integration profiles and risk of insertional mutagenesis. 30 The second is the identification of the optimal hematopoietic cell types for expression of either FVIII or FIX. The third is the development of protocols and agents designed to minimize transplantation conditioning regimen-related toxicity while maintaining sufficient and durable HSC engraftment.…”