Unique Integration Profiles of Gammaretrovirus, Lentivirus, and Foamy Virus Transduced Dog Long-Term Repopulating Cells.
Authors: Keyser Kirsten A. , Beard Brian C. , Trobridge Grant , Peterson Laura J. , Miller Dan , Jacobs Mike , Kaul Rajinder , Kiem Hans-Peter 
Published: October 2019

Abstract
Retroviral vectors have been the most effective gene delivery vehicles for hematopoietic stem cell (HSC) gene therapy and patients are now being successfully treated for genetic diseases. Following the success of gene therapy for inherited disorders, such as X-linked severe combined immunodeficiency (SCID), three of the patients developed overt leukemia, and a spontaneous expansion of gene-marked cells has been described in two patients treated in an X-linked chronic granulomatous clinical trial. In spite of these outcomes clinical gene therapy trials continue to show efficacy for patients with genetic diseases. Valuable data has been generated regarding retrovirus integration profiles and factors that can lead to clonal dominance (i.e. enhancer activation and multiple integration sites) in murine models. Large animal studies extend these analyses to clinically predictive models, that should more accurately indicate what would occur in human clinical trials.
We analyzed the retrovirus integration profile in dogs transplanted with cells gene-modified with either gammaretrovirus (n=5 dogs), HIV-derived lentivirus (n=6 dogs), or foamy virus (n=2 dogs) vectors using a sensitive LAM-PCR method modified to facilitate high-throughput analysis. The samples used for LAM-PCR ranged from 95–600 days after transplantation. We analyzed over 14,500 sequence reads and were able to unambiguously align a total of 555 unique integration sites to the dog genome with 82 unique gammaretroviral integrants, 210 unique lentiviral integrants, and 263 unique foamy viral integrants. We defined the integration patterns relative to transcription units, a subset of previously defined proto-oncogenes and CpG islands. The most prevalent clustering within 50 kilobases of RefSeq transcription start sites and into CpG islands was seen in gammaretroviral integrants (73.2% and 3.7%, respectively) and to a lesser extent in foamy viral integrants (53.6% and 3.4%, respectively). Regarding integration into RefSeq genes, lentiviral integrants showed the most significant increase (58.6%) relative to random integration analysis (37.3%). Gammaretroviral integrants were also significantly increased both in proto-oncogenes (7.3%) and within 50 kilobases of the transcription start site of proto-oncogenes (7.3%). In addition, even though fewer sites have been analyzed and localized for gammaretrovirus vectors, compared to lentiviral and foamy viral sites, we found two distinct gammaretroviral integrants in the MDS/Evi1 locus with no lentiviral or foamy viral integrants localized in this region.
While no single factor distinguishes one of the retroviruses as the ‘safest’ vector system, the low frequency of integration of foamy virus into RefSeq genes and the decreased density of lentiviral integrants around transcription start sites suggest that these vectors may be preferred relative to gammaretroviral vectors. Additionally, high titer self-inactivating (SIN) vector design has been achieved using both lentivirus and foamy virus which should decrease the risk of enhancer activation relative to intact LTR constructs. These aspects and others, including weaker/regulated internal promoters, and chromatin insulators are important factors that should be considered when designing vectors for clinical gene therapy applications.