title: Cassette deletion in multiple shRNA lentiviral vectors for HIV-1 and its impact on treatment success.
abstract: BACKGROUND
  Multiple short hairpin RNA (shRNA) gene therapy strategies are currently being investigated for treating viral diseases such as HIV-1. It is important to use several different shRNAs to prevent the emergence of treatment-resistant strains. However, there is evidence that repeated expression cassettes delivered via lentiviral vectors may be subject to recombination-mediated repeat deletion of 1 or more cassettes.
  
  
  RESULTS
  The aim of this study was to determine the frequency of deletion for 2 to 6 repeated shRNA cassettes and mathematically model the outcomes of different frequencies of deletion in gene therapy scenarios. We created 500+ clonal cell lines and found deletion frequencies ranging from 2 to 36% for most combinations. While the central positions were the most frequently deleted, there was no obvious correlation between the frequency or extent of deletion and the number of cassettes per combination. We modeled the progression of infection using combinations of 6 shRNAs with varying degrees of deletion. Our in silico modeling indicated that if at least half of the transduced cells retained 4 or more shRNAs, the percentage of cells harboring multiple-shRNA resistant viral strains could be suppressed to < 0.1% after 13 years. This scenario afforded a similar protection to all transduced cells containing the full complement of 6 shRNAs.
  
  
  CONCLUSIONS
  Deletion of repeated expression cassettes within lentiviral vectors of up to 6 shRNAs can be significant. However, our modeling showed that the deletion frequencies observed here for 6x shRNA combinations was low enough that the in vivo suppression of replication and escape mutants will likely still be effective.
authors.affiliation: Johnson and Johnson Research Pty Ltd, Level 4 Biomedical Building, Australian Technology Park, Eveleigh, NSW 1430, Australia. glen@madebyglen.com
authors.forename: Glen J
authors.initials: GJ
authors.lastname: Mcintyre
authors.forename: Yi-Hsin
authors.initials: YH
authors.lastname: Yu
authors.forename: Anna
authors.initials: A
authors.lastname: Tran
authors.forename: Angel B
authors.initials: AB
authors.lastname: Jaramillo
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Arndt
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Millington
authors.forename: Maureen P
authors.initials: MP
authors.lastname: Boyd
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Elliott
authors.forename: Sylvie W
authors.initials: SW
authors.lastname: Shen
authors.forename: John M
authors.initials: JM
authors.lastname: Murray
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Applegate
mesh_terms: D003198:Computer Simulation; D015316:Genetic Therapy / Q000379:methods*; D015658:HIV Infections / Q000628:therapy*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*; D034741:RNA, Small Interfering / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D034741:RNA, Small Interfering