rDNA-directed integration by an HIV-1 integrase—I-PpoI fusion protein

Abstract

Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.

Development of Lentiviral Vectors for Targeted Integration and Protein Delivery

“…For example, for HIV-1 based vectors, it has been demonstrated that by modifying the DNA-binding capabilities of the IN cofactor protein LEDGF/p75, native LV integration preferences can be altered [7][8][9]. We have also shown that LV integration into a predetermined site can be increased significantly with the aid of IN-fusion proteins [10].…”

“…We took IN-fusion proteins into modern third-generation LVs and showed that they could be used for the delivery of desired proteins into transduced cell nuclei [13]. Moreover, such vectors were able to enrich vector integration in predetermined genomic safe harbor target sites when IN was fused to a meganuclease recognizing a sequence at these sites [10]. This chapter describes the original design of IN-fusion protein-encoding constructs which drive the packaging of the proteins into thirdgeneration LV particles that can be used to study targeted integration or protein transduction.…”

Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity

“…Correct packaging of the IN-I-PpoI H78A into LVVs was verified by immunoblotting using an antibody against HIV-1 IN, as described previously [10]. VSV-G pseudotyped third generation LVVs were produced, concentrated, and titered with p24 ELISA and flow cytometry as described [8].…”

Delivering the Goods for Genome Engineering and Editing

“…Due to the wealth of recognition sites and robust lentiviral transfer of the protein cargo, delivery of I-PpoI was found to be cytotoxic in a manner dependent on the enzyme activity. 111,112 However, by fusing the integrase to a mutated catalytically inactive I-PpoI variant, it was possible to promote targeted lentiviral insertions into ribosome RNA genes. 111 It is important to note also that vectors carrying an integrase fusion protein maintained the capacity to transfer vector RNA.…”

“…111,112 However, by fusing the integrase to a mutated catalytically inactive I-PpoI variant, it was possible to promote targeted lentiviral insertions into ribosome RNA genes. 111 It is important to note also that vectors carrying an integrase fusion protein maintained the capacity to transfer vector RNA. 110 Our own endeavors to develop lentiviral protein transduction for genome engineering were directed first at incorporating the hyperactive piggyBac transposase hyPBase 113 in lentiviral particles.…”