A Fourth Generation Lentiviral Vector: Simplifying Genomic Gymnastics
Authors: Ben Berkhout
Journal: Molecular Therapy (2017)

Lentiviral vectors have become a popular tool for gene transfer experiments and gene therapy approaches, particularly when stable gene transfer is required. They allow durable transgene expression because the vector integrates into the host cell genome. However, depending on the specific application, difficulties have persisted with respect to the efficiency and safety of this vector system. The article by Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar in this issue of Molecular Therapy describes a new lentiviral vector termed LTR1 that is both more efficient and safer than currently employed versions. This was achieved by reconfiguration of the genetic elements that control the complicated replication process of the vector. Lentiviral vectors are based on the genome of the HIV type 1 (HIV-1) or that of other lentiviruses, a subclass of the retrovirus family that employs a rather convoluted mechanism for the copying of its RNA genome. Retroviruses encode a unique reverse transcriptase enzyme that copies the viral RNA genome into DNA. It took quite a few years to dissect the intricate reverse transcription process, which employs a cellular transfer RNA (tRNA) molecule as primer and requires two mandatory strand transfers ("jumps") in order to generate a double-stranded DNA (dsDNA) copy that is, in fact, larger than the input RNA genome (Figure 1, left), and carrying two long terminal repeats (LTRs).2Gilboa E. Mitra S.W. Goff S. Baltimore D. A detailed model of reverse transcription and tests of crucial aspects.Cell. 1979; 18: 93-100Abstract Full Text PDF PubMed Scopus (420) Google Scholar This genome expansion is critical for virus reproduction because it regenerates full-length 5′LTR sequences that act as the transcriptional promoter for synthesis of new viral RNA transcripts in the next round of infection. Knowledge of this replication mechanism informed the design of retroviral and, subsequently, lentiviral vectors. The RNA genome needs to carry all cis-acting signals/sequences that control RNA packaging into virion particles, subsequent reverse transcription into DNA, and, finally, integration into the host genome. From that stably integrated position, the therapeutic transgene is usually expressed from the endogenous 5′LTR promoter. The essential viral proteins (Gag, Pol, Tat, Rev) are produced in trans by a vector-producing cell, and the Envelope protein is replaced by the more stable VSV-G protein, which allows entry into more cell types. To improve safety, further adaptations were made by removing additional HIV-1 sequences, such as the use of an exogenous promoter for transgene expression, which also allowed removal of sequences encoding the viral Tat transcriptional activator. Current third generation lentiviral vectors encode only three of the nine HIV-1 proteins (Gag, Pol, Rev), which are expressed from separate plasmids to avoid recombination-mediated generation of a replication-competent virus. As an additional safety feature, heavily mutated codon-optimized versions of these "packaging constructs" can be used. To avoid insertional oncogene activation, the 3′LTR promoter was inactivated by a deletion that is inherited during the reverse transcription process in both the 5′ and 3′LTRs, thereby "self-inactivating" both viral promoter elements.3Hacein-Bey-Abina S. Garrigue A. Wang G.P. Soulier J. Lim A. Morillon E. Clappier E. Caccavelli L. Delabesse E. Beldjord K. et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J. Clin. Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar Overall, this vector encodes approximately 1.7 kb (or 17.5%) of the complete 9.7 kb HIV-1 genome sequence.4Zufferey R. Dull T. Mandel R.J. Bukovsky A. Quiroz D. Naldini L. Trono D. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J. Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar With an exogenous promoter of around 0.5 kb, this leaves a "transgene packaging capacity" of approximately 8.8 kb. Many minor, but no major, improvements have been made since then to the basic design of the third-generation lentiviral vector. Previous attempts to further minimize the lentiviral vector genome failed.5Cui Y. Iwakuma T. Chang L.-J. Contributions of viral splice sites and cis-regulatory elements to lentivirus vector function.J. Virol. 1999; 73: 6171-6176PubMed Google Scholar, 6Delviks K.A. Hu W.S. Pathak V.K. Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.J. Virol. 1997; 71: 6218-6224Crossref PubMed Google Scholar, 7Fang Y. Gong X. Xu M. Zeng F. Zhang J. A self-deletion lentiviral vector to reduce the risk of replication-competent virus formation.J. Gene Med. 2013; 15: 102-112Crossref PubMed Scopus (6) Google Scholar Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar show that one can redesign a lentiviral vector by placing several essential RNA signals (PBS-Ψ-RRE) outside the viral backbone; more specifically, downstream of the 3′LTR (Figure 1, right). These signals are present in the viral RNA during vector production when they are needed, but they are subsequently lost during reverse transcription and, thus, are not copied into the transduced transgene, thus increasing the safety. But let us first discuss the vector reconfiguration that was meant to streamline the reverse transcription process. Wild-type HIV-1 (Figure 1, left) initiates reverse transcription very close to the 5′ end of the RNA genome by binding of a specific cellular tRNA primer to the primer binding site (PBS in Figure 1). Initiation of reverse transcription is a complex well-regulated process that also requires an upstream primer activation signal (PAS),8Beerens N. Berkhout B. The tRNA primer activation signal in the HIV-1 genome is important for initiation and processive elongation of reverse transcription.J. Virol. 2002; 76: 2329-2339Crossref PubMed Scopus (76) Google Scholar which is encoded within the upstream LTR sequences. Because, initially, only a short copy DNA (cDNA) of the 5′ end of the genome is produced, transfer of the partial cDNA to repeated LTR sequences in the 3′ end of the viral RNA genome is required (first-strand transfer), followed by further copying of the RNA genome. Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar removed the 5′LTR, which incapacitates reverse transcription by removal of the PAS motif, and placed a functional primer binding site immediately downstream of the PAS-encoding 3′LTR. This priming position allows the generation of a full-length cDNA without the complex first-strand transfer. Both wild-type HIV-1 and the novel PBS1 vector require another strand transfer (second or first, respectively) to initiate second-strand DNA synthesis and formation of the integration-competent dsDNA. As noted, the LTR1 design allowed Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar to move multiple RNA signals (PBS-Ψ-RRE) outside the vector backbone. This includes the Rev-responsive element (RRE) that facilitates Rev-mediated nuclear export of the transcript, the psi or the Ψ signal that mediates packaging of the RNA into viral particles, and some splice signals. This further minimizes HIV-derived sequences in the transduced transgene to just 441 bp (just 4.8%), which includes critical LTR domains, the primer binding site, and PAS elements to regulate initiation of first-strand synthesis during reverse transcription8Beerens N. Berkhout B. The tRNA primer activation signal in the HIV-1 genome is important for initiation and processive elongation of reverse transcription.J. Virol. 2002; 76: 2329-2339Crossref PubMed Scopus (76) Google Scholar, 9Das A.T. Klaver B. Berkhout B. Reduced replication of human immunodeficiency virus type 1 mutants that use reverse transcription primers other than the natural tRNA(3Lys).J. Virol. 1995; 69: 3090-3097PubMed Google Scholar and the polypurine tracks that control second-strand DNA synthesis.10Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap.Cell. 2000; 101: 173-185Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar The LTR1 design sustained higher transgene expression than a standard lentiviral vector in vivo. Perhaps more intriguingly, transgene expression was initiated much earlier from the LTR1 vector. As this coincides with an earlier appearance of the reverse transcription products, it is likely explained by the significant simplification of the reverse transcription mechanism (from two to one jump). Indeed, it is well-known that reverse transcription of the HIV-1 genome takes a considerable time in infected T cells.11O'Brien W.A. Namazi A. Kalhor H. Mao S.H. Zack J.A. Chen I.S. Kinetics of human immunodeficiency virus type 1 reverse transcription in blood mononuclear phagocytes are slowed by limitations of nucleotide precursors.J. Virol. 1994; 68: 1258-1263PubMed Google Scholar It is therefore important to lower this hurdle for lentiviral vectors, which was done by minimization of the size and complexity (only one strand transfer) of the template. The minimized LTR1 vector design offers additional advantages: improved safety because of the reduced ability to be remobilized into HIV-1 particles in vector-transduced cells and fewer erroneous trans-splicing events into human transcripts. I propose to call this LTR1 design a fourth generation lentiviral vector, although further improvement of the vector titer would be welcome. Perhaps a philosophical issue, but one wonders why HIV-1 and other retroviruses did not evolve this seemingly more efficient shortcut of the reverse transcription process as executed by the designer LTR1 vector? The simple answer is that the LTR1 vector is perfect for transgene delivery, but it does, in fact, not regenerate a copy of the vector genome that includes two complete flanking LTR motifs. In other words, the LTR1 design is great for single-cycle gene transduction, exactly what we want in a gene therapy setting, but it cannot be utilized by a replicating viral entity. As such, use of the word "life-cycle" seems inappropriate for the PBS1 design (see Figure 1 in Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Vink et al.1Vink C.A. Counsell J.R. Perocheau D.P. Kards R. Buckley S.M.K. Brugman M.H. Galla M. Schambach A. McKay T.R. Waddington S.N. Howe S.J. Eliminating HIV-1 packaging sequences from lentiviral vector proviruses enhances safety and expedites gene transfer for gene therapy.Mol Ther. 2017; 25 (this issue): 1790-1804Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar demonstrate that the LTR1 vector can be produced at titers sufficient for preclinical gene therapy in a hemophilia B mouse model. Gene therapy successes with lentiviral vectors were recently described (e.g., for X-linked adrenoleukodystrophy and the Wiskott-Aldrich syndrome).12Aiuti A. Biasco L. Scaramuzza S. Ferrua F. Cicalese M.P. Baricordi C. Dionisio F. Calabria A. Giannelli S. Castiello M.C. et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (809) Google Scholar, 13Cartier N. Hacein-Bey-Abina S. Bartholomae C.C. Veres G. Schmidt M. Kutschera I. Vidaud M. Abel U. Dal-Cortivo L. Caccavelli L. et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1177) Google Scholar Perhaps most importantly, safety of the current lentiviral vectors was demonstrated, although these trials were performed with a small number of patients. The new LTR1 vector design should further spur the clinical use of these vectors, e.g., by allowing more sizeable transgene inserts. This is particularly important for contemporary CRISPR/Cas approaches, especially when multiple guide RNAs must be expressed on top of the sizeable Cas endonuclease.14Wang G. Zhao N. Berkhout B. Das A.T. A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures.Cell Rep. 2016; 17: 2819-2826Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene TherapyVink et al.Molecular TherapyMay 24, 2017In BriefCounsell and colleagues have developed a novel lentiviral vector, LTR1, designed to minimize the incorporation of HIV-1 sequences during transduction. Their data show that LTR1 potentially offers improved safety in gene therapy, while demonstrating robust gene therapy efficacy in correcting a mouse model of hemophilia B. Full-Text PDF Open Access