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

Abstract

Retroviral vectors have shown their curative potential in clinical trials correcting monogenetic disorders. However, therapeutic benefits were compromised due to vector-induced dysregulation of cellular genes and leukemia development in a subset of patients. Bromodomain and extraterminal domain (BET) proteins act as cellular cofactors that tether the murine leukemia virus (MLV) pre-integration complex to host chromatin via interaction with the MLV integrase (IN) and thereby define the typical gammaretroviral integration distribution. We engineered next-generation BET-independent (Bin) MLV vectors to retarget their integration to regions where they are less likely to dysregulate nearby genes. We mutated MLV IN to uncouple BET protein interaction and fused it with chromatin-binding peptides. The addition of the CBX1 chromodomain to MLV INW390A efficiently targeted integration away from gene regulatory elements. The retargeted vector produced at high titers and efficiently transduced CD34+ hematopoietic stem cells, while fewer colonies were detected in a serial colony-forming assay, a surrogate test for genotoxicity. Our findings underscore the potential of the engineered vectors to reduce the risk of insertional mutagenesis without compromising transduction efficiency. Ultimately, combined with other safety features in vector design, next-generation BinMLV vectors can improve the safety of gammaretroviral vectors for gene therapy.

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

“…This could be studied using the BET-independent gammaretroviral vectors that integrate outside the active TSS and enhancers [ 20 , 21 ]. In this context, a recent study [ 22 ] reported that the next-generation BET-independent MLV-derived vectors showed the same stability of expression without genotoxicity when compared to a wild-type MLV-derived vector.…”

“…Disruption of the interaction between the intasome and BET proteins or LEDGF/p75 has resulted in the decreased efficiency of retrovirus integration and retargeting outside the preferred genomic regions [ 12 , 20 ]. Hence, retargeting strategies are already being used to design the next generation of retroviral vectors [ 20 , 21 , 22 ] that demonstrate the decreased risk of cellular protooncogene transactivation [ 23 ]. In addition to genetic manipulation of retroviral integrase, specific HIV-1 integrase-LEDGF/p75 interaction can also be allosterically inhibited with small molecules of LEDGF/p75 inhibitors (LEDGINs), which represents a new and promising therapeutic strategy [ 24 ].…”

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

“…Our results indicate that removing the TP was insufficient to redirect all integrations away from active promoters and strong enhancers, or to eliminate the stochastic events that can select for oncogenic activation. Ultimately, a modified vector that combines SIN LTRs to eliminate strong viral enhancers [10,81] with insertions/replacement of the IN TPto redirect integration to less active regions [39,82] could decrease vector genotoxicity and overcome current limitations for clinical applications.…”

“…Subsequent approaches involving self-inactivating (SIN) vectors [38] or lentiviral vectors have been used to address these outcomes [30]. Alternatively, addressing the integration target-site bias of gammaretroviruses to integrate preferentially at promoter/enhancer regions by altering or eliminating their interaction with host BET protein could alter the oncogenic potential of these vectors [4,39].…”

Shifting Retroviral Vector Integrations Away from Transcriptional Start Sites via DNA-Binding Protein Domain Insertion into Integrase

“… 10 , 11 , 12 , 13 , 14 , 16 , 18 Based on these findings, several trials, often involving viral integrase engineering, have been conducted to alter the retroviral integration patterns by interfering with the interactions between the cellular tethering factors and the virus, or by conferring viruses the ability to interact with new host proteins. 20 , 21 , 22 , 23 In particular, MLV integrations around TSSs have been successfully reduced, but still occur at significantly high frequencies, indicating that there might be other, unknown mechanisms underlying the integration process. 20 However, searching the complete MLV integration mechanisms and finding ways to block all the critical mechanisms require substantial time and effort.…”