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Integrase-Defective Lentiviral Vectors: Progress and Applications

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Integrase-Defective Lentiviral Vectors: Progress and Applications Gene Therapy (2010) 17, 150–157 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 $32.00 www.nature.com/gt REVIEW Integrase-defective lentiviral vectors: progress and applications MB Banasik1,2 and PB McCray Jr1,2 1Department of Pediatrics, Program in Gene Therapy, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA and 2Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA Lentiviral vectors (LVs) offer the advantages of a large in which transient gene expression is desired. Several recent packaging capacity, broad cell tropism or specific cell-type publications outline the development and initial biological targeting through pseudotyping, and long-term expression characterization of such vectors. Here, we discuss the poten- from integrated gene cassettes. However, transgene integ- tial applications and new directions for the development of ration carries a risk of disrupting gene expression through integration-defective LVs. insertional mutagenesis and may not be required for all Gene Therapy (2010) 17, 150–157; doi:10.1038/gt.2009.135; applications. A non-integrating LV may be beneficial in cases published online 22 October 2009 Keywords: HIV; FIV; episome; zinc-finger nuclease; vaccine Introduction necessary for integration.9,10,14 IN then facilitates strand transfer through a one-step mechanism that both induces Lentiviral vectors (LV) are an important class of gene a staggered break in the phosphodiester bonds of the transfer vectors. LV have a large packaging capacity target DNA and joins a single vector DNA end to the (410 kb transgene cassette1,2) and envelope glycoprotein target DNA through a transesterification reaction.11,14,15 pseudotyping achieves broad or specific tropism as Once both vector DNA ends are attached, host enzymes necessary. These vectors are also associated with low repair the resulting gapped intermediate, generating an immunogenicity. Importantly, they transduce mitotically integrated provirus (Figure 2).10,14,16 quiescent cell types, including many relevant targets for Although integration is generally described as the gene transfer. One of the greatest benefits of LVs is their specific end point of lentiviral gene transfer, DNA ability to integrate a transgene copy into target cell episomes are also generated by lentiviruses and their chromosomes, allowing for long-term expression. How- derived vectors.15,17,18 Among these, the linear DNA ever, this process carries finite risks of detrimental episome is the precursor to the integrated provirus insertional mutagenesis, prompting examination of (Figure 2).11,15,19 Two types of circular episomes with intact alternatives to vector-mediated integration. viral coding regions are also produced. Homologous recombination (HR) within the LTRs generates a circular episome with a single LTR (1-LTR circle).15,17 In addition, LV transduction biology ligation of nicks in the circular DNA intermediates during the final stage of reverse transcription also results in 1-LTR After vector internalization and reverse transcription in circle episome formation.15,20 Non-homologous end joining the cytoplasm, the vector double-stranded DNA is of the linear episome results in a circular episome with two incorporated into the pre-integration complex. A critical adjacent LTRs (2-LTR circle) (Figure 2).15,17,21 Generally, component of the integration complex is viral integrase 1-LTR circles are more prevalent than 2-LTR circles, but (IN), a 32–45 kDa protein,3–7 which catalyzes viral DNA ratios vary.17,18,22–24 Both 1- and 2-LTR episomes can express integration into the host genome (Figure 1).8–10 Once the proteins because the genome or transgene remains intact.25,26 integration complex reaches the nucleus, IN mediates IN can also auto-integrate within the viral DNA itself, but integration between vector and host DNA. the disruption of the viral coding region generally renders Before nuclear translocation,4,11,12 IN catalyzes the the resulting products inactive.17,27 removal of two nucleotides from the 30 end of the Episomes do not persist in dividing cells because they reverse-transcribed genome at an invariant CA dinucleo- are not integrated and lack an origin of replication (ORI). tide.9,10,13 This 30-processing forms hydroxyl groups It was originally hypothesized that 1- and 2-LTR episomes were rapidly degraded as shown by PCR Correspondence: Dr PB McCray Jr, Department of Pediatrics, 240G analysis of 2-LTR circles present in peripheral blood EMRB, Carver College of Medicine, The University of Iowa, Iowa mononuclear cells from HIV infected patients.18 How- City, IA 52242, USA. ever, subsequent studies determined that the apparent E-mail: [email protected] half-life of these episomes correlated with the rate of cell Received 13 June 2009; revised 16 September 2009; accepted 18 23,28 September 2009; published online 22 October 2009 division. Therefore, viral episomes are stable and the Integrase-defective LVs: progress and applications MB Banasik and PB McCray Jr 151 Figure 1 Diagram of HIV IN. A schematic representation of the three domains of IN. Sites of IDLV mutations are indicated by arrows. Mutations affecting the catalytic triad are in bold. Mutations affecting genomic DNA binding are underlined. Other IDLV mutations are italicized. cancer. Retroviral integration site selection was initially hypothesized to be random, based largely on in vitro studies.6,32 However, the risks of insertional mutagenesis were highlighted during the X-SCID retroviral gene therapy trials.33–37 Unexpectedly, five patients developed T-cell such as leukemia as a result of vector treat- ment.33,34,36,37 According to the model of site selection at the time, multiple oncogenic events should have been exceedingly rare. Extensive integration site mapping studies subsequently showed that retroviral integration site selection is not random and varies between gammaretroviruses, lentiviruses and other classes of integrating viral and non-viral vectors. Studies show that gammaretroviruses preferentially integrate in 50 flanking regions of genes near transcrip- tion start sites, increasing the possibility of affecting nearby gene expression.32 Unlike gammaretroviruses, LV do not exhibit an increased integration site preference for 50 flanking regions and avoid CpG islands, a character- istic feature found near transcription start sites.32 Instead, this vector class integrates across the entire transcrip- tional unit.32,38–41 A recent study showed that LV with chimeric gammaretroviral LTRs were still significantly less genotoxic than gammaretroviral vectors.42 Therefore, LV site selection preferences are distinct from gammaretroviral vectors and may be less genotoxic for therapeutic uses. Though the probability of aberrant gene activation or disruption are less likely after LV integration, insertional mutagenesis risks must be considered for clinical applications.2,30,33,34,43 One approach to circumvent detrimental insertional Figure 2 Representation of integration process. Integration begins 0 mutagenesis risks is the generation of a vector with with IN multimerization and vector DNA binding. LTR 3 cleavage properties of specific ‘safe’ site integration.44,45 However, generates hydroxyl groups necessary for the catalytic process. IN binds the host genomic DNA and integration occurs. A variety of mutations permanent transgene expression is not necessary or block different steps of this process, indicated below each stage, desirable in all applications. An alternative to a perma- rendering the vector IN defective. This prevents provirus formation nently integrated transgene is transient expression from and vector DNA remains as one of three primary episome types. an integration-defective LV vector (termed IDLV, also INdef, IN- or NILV). Non-integrating gammaretroviral vectors have been described,46 but because they cannot observed loss occurred from dilution of the episomes transduce non-dividing cells, several clinically relevant during cell replication.23,28 cell types are not effectively targeted. However, LV transduce slowly dividing cell types; therefore, episomal vector DNA is diluted slowly and persists.47–49 Several LV integration reports have detailed the production of HIV IDLVs.13,22,44,47–60 In addition, feline immunodeficiency As LVs introduce a permanent transgene copy into the virus (FIV) IDLVs are also described.48,61 host genome, they achieve persistent gene expres- sion.15,19,29–31 This may correct a monogenic recessive disorder. However, LV integration is inherently muta- IDLV mutations genic and could induce detrimental side effects depend- ing on the specific host gene disrupted. For instance, the IN catalyzes LV integration, with HIV IN the most activation of a protooncogene or disruption of a tumor extensively studied. IN has three separate protein suppressor gene could lead to the development of domains as determined by proteolysis and functional Gene Therapy Integrase-defective LVs: progress and applications MB Banasik and PB McCray Jr 152 studies (Figure 1).12,62 The N-terminal domain contains from non-integrating lentiviral infections26,72,76 and other an HH-CC zinc-finger binding domain that primarily non-integrating viruses. binds viral DNA5,63,64 and facilitates required IN After confirmation of lentiviral episomal persis- multimerization.65,66 The C-terminal domain is the least tance,23,28 the potential of IDLVs was further examined. conserved retroviral IN domain5,67,68 and binds
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