Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

Abstract

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

Engineering Next-Generation BET-Independent MLV Vectors for Safer Gene Therapy

“…Previously, we re-engineered the MLV- and HIV-cellular tethering cofactors (BET and LEDGF/p75, respectively) and demonstrated efficient redistribution of retroviral integration without compromising transgene expression. 29 , 63 , 64 , 65 However, this approach requires the introduction (at least transient) of artificial anchors in target cells prior to application of the therapeutic vectors, 63 , 64 which is not always desirable in a clinical setting. A more straightforward strategy is to directly engineer vector particles to contain proteins with adapted or unique binding domains to direct integration.…”

Cellular and molecular mechanisms of HIV-1 integration targeting

“…JPO2) and MLL in proximity to chromatin [106], and thus HIV-1 hijacks this ancient chromatin-associated molecular beacon to fulfill its nefarious needs. Hybrid LEDGF/p75 constructs that swapped the N-terminal chromatin binding elements for heterologous chromatin readers supported HIV-1 infection [107] and redirected integration to novel positions in the genome that were consistent with the known chromatin binding properties of the substituted domains [108–111]. The plasticity of this approach was rather remarkable, as both promoter-proximal readers such as PHD (plant homeodomain) fingers as well as heterochromatin protein modules such as CBX1 and HP1 similarly supported HIV-1 infection.…”

“…LEDGF/p75 is inefficiently packaged into HIV-1 particles and the scant amount that is packaged is cleaved by the viral protease [113], so the potential to hitchhike retargeting LEDGF/p75 into the cell as a virus vector component is an apparent non-starter. The need to introduce a hybrid construct into patient cells prior to a therapeutic lentiviral vector necessarily complicates the clinical utility of retargeting LEDGF/p75 constructs in human gene therapy [103,104,111,114].…”

Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes

“…LEDGF/p75 can be truncated by deleting the N-terminal chromatin-reading PWWP-domain, and replacing this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells can result in more randomly distributed lentiviral integration throughout the host-cell genome [ 22 ].…”