Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Abstract

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

CAR-Expressing Natural Killer Cells for Cancer Retargeting

“…The authors argued that many factors influence transduction efficacy (e.g., transduction method and codon optimisation of the construct) and speculated that primary NK cells may exert specific responses against viral particles of certain pseudotypes [22]. Moreover, an advanced alpha-retroviral system was used in these works, which might tip the balance in favour of this approach [22, 25, 26]. However, it would be too early to speculate that different retroviral properties and elicited anti-viral responses correlate with the retrovirus-specific differences in transduction efficiencies, with RD114/TR pseudotyped alpha-retroviral particles, and with improved gene transfer in NK cells.…”

Development of gene therapy for blood disorders: an update

“…20 A split-packaging design for self-inactivating a-retroviral vectors has been developed and shown to be capable of generating high-titer vector particles. 19 The therapeutic potential for this class of vectors has been shown by functional correction of chronic granulomatous disease (CGD) in a cell line as well as within transduced human cells that engraft in the mouse model.…”

“…18 The a-retroviral vectors are being considered for therapeutic gene transfer as well. [19][20][21] Such vectors integrate more uniformly within the genome, particularly within intergenic regions compared with g-retroviral vectors, which are found near transcriptional start sites and cytosine guanine dinucleotide islands, and lentiviral vectors, which tend to integrate within genes. 22 This favorable integration pattern has been shown to result in lower genotoxicity compared with lentiviral and g-retroviral vectors.…”

Alpharetroviral Self-inactivating Vectors: Long-term Transgene Expression in Murine Hematopoietic Cells and Low Genotoxicity

“…6,12 Therefore, we have developed alpharetroviral self-inactivating (SIN) vectors, eliminating enhancer and promoter elements from the LTRs, and set up an advanced splitpackaging system, additionally removing viral-coding sequences and retroviral splice sites from the vector. 13 In the present study, we performed side-by-side comparisons of alpharetroviral, gammaretroviral, and lentiviral SIN vectors in a serial murine bone marrow (BM) transplantation model, which allowed us to evaluate the potential of alpharetroviral SIN vectors in relation to state-of-the-art clinically used vectors. We analyzed transgene expression levels, mean vector copy numbers (mVCNs), and integration site distributions in primary and secondary recipients (for a period of up to 31 and 17 weeks, respectively).…”