A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors.

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5' leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5-gag interaction and a more stable structure with a U5-AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5' region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

Hybrid lentiviral vectors.

Lentiviral vectors have remarkable cell entry and gene delivery properties that make them highly attractive for gene therapy. However, all integration-competent gene delivery systems have come under scrutiny for possible adverse insertional events. Circumventing the risk of insertional mutagenesis, integration-deficient human immunodeficiency virus (HIV)-1-derived vectors have been shown to support durable transcription of transgenes in certain nonmitotic cell lineages. In mitotic cell populations, such nonintegrated viral forms are lost during cell division and so have time-limited effects. Hybrid lentiviral vectors that harness the cell entry properties of HIV to facilitate carriage of alternative DNA modification systems into cells may allow durable genetic modification with more favorable integration profiles. Thus, systems, which have previously been plasmid-based such as those based on nuclease-enhanced homologous recombination (HR) and artificial transposons, have been incorporated into the viral genome to allow them to "hitch-hike" into cells that are difficult to transfect. Here, we review recent progress in the development of such hybrid lentiviral systems and consider potential applications of such vectors.

The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre.

Lentiviral vectors are showing success in the clinic, but producing enough vector to meet the growing demand is a major challenge. Furthermore, next-generation gene therapy vectors encode multiple genes resulting in larger genome sizes, which is reported to reduce titers. A packaging limit has not been defined. The aim of this work was to assess the impact of genome size on the production of lentiviral vectors with an emphasis on producer cell mRNA levels, packaging efficiency, and infectivity measures. Consistent with work by others, vector titers reduced as genome size increased. While genomic infectivity accounted for much of this effect, genome sizes exceeding that of clinical HIV-1 isolates result in low titers due to a combination of both low genomic infectivity and decreased packaging efficiency. Manipulating the relative level of genomic RNA to gag-pol mRNA in the producer cells revealed a direct relationship between producer cell mRNA levels and packaging efficiency yet could not rescue packaging of oversized genomes, implying a de facto packaging defect. However, independent of genome size, an equimolar ratio between wild-type gag-pol mRNA and vector genomic RNA in producer cells was optimal for titer.

HIV-1 sequences in lentiviral vector genomes can be substantially reduced without compromising transduction efficiency.

Many lentiviral vectors used for gene therapy are derived from HIV-1. An optimal vector genome would include only the viral sequences required for transduction efficiency and gene expression to minimize the amount of foreign sequence inserted into a patient's genome. However, it remains unclear whether all of the HIV-1 sequence in vector genomes is essential. To determine which viral sequences are required, we performed a systematic deletion analysis, which showed that most of the gag region and over 50% of the env region could be deleted. Because the splicing profile for lentiviral vectors is poorly characterized, we used long-read sequencing to determine canonical and cryptic splice site usage. Deleting specific regions of env sequence reduced the number of splicing events per transcript and increased the proportion of unspliced genomes. Finally, combining a large deletion in gag with repositioning the Rev-response element downstream of the 3' R to prevent its reverse transcription showed that 1201 nucleotides of HIV-1 sequence can be removed from the integrated vector genome without substantially compromising transduction efficiency. Overall, this allows the creation of lentiviral vector genomes that contain minimal HIV-1 sequence, which could improve safety and transfer less viral sequence into a patient's DNA.

Minimal impact of ZAP on lentiviral vector production and transduction efficiency.

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

Sleeping beauty transposition from nonintegrating lentivirus.

Lentiviral vectors enter cells with high efficiency and deliver stable transduction through integration into host chromosomes, but their preference for integration within actively transcribing genes means that insertional mutagenesis following disruption of host proto-oncogenes is a recognized concern. We have addressed this problem by combining the efficient cell and nuclear entry properties of HIV-1-derived lentiviral vectors with the integration profile benefits of Sleeping Beauty (SB) transposase. Importantly, this integration enzyme does not exhibit a preference for integration within active genes. We generated integrase-deficient lentiviral vectors (IDLVs) to carry SB transposon and transposase expression cassettes. IDLVs were able to deliver transient transposase expression to target cells, and episomal lentiviral DNA was found to be a suitable substrate for integration via the SB pathway. The hybrid vector system allows genomic integration of a minimal promoter-transgene cassette flanked by short SB inverted repeats (IRs) but devoid of HIV-1 long terminal repeats (LTRs) or other virus-derived sequences. Importantly, integration site analysis revealed redirection toward a profile mimicking SB-plasmid integration and away from integration within transcriptionally active genes favored by integrase-proficient lentiviral vectors (ILVs).

Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency.

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Erratum: Lentiviral vectors can be used for full-length dystrophin gene therapy.

This corrects the article DOI: 10.1038/srep44775.

Lentiviral vectors can be used for full-length dystrophin gene therapy.

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Lentiviral vectors can be used for full-length dystrophin gene therapy.

Duchenne Muscular Dystrophy (DMD) is caused by a lack of dystrophin expression in patient muscle fibres. Current DMD gene therapy strategies rely on the expression of internally deleted forms of dystrophin, missing important functional domains. Viral gene transfer of full-length dystrophin could restore wild-type functionality, although this approach is restricted by the limited capacity of recombinant viral vectors. Lentiviral vectors can package larger transgenes than adeno-associated viruses, yet lentiviral vectors remain largely unexplored for full-length dystrophin delivery. In our work, we have demonstrated that lentiviral vectors can package and deliver inserts of a similar size to dystrophin. We report a novel approach for delivering large transgenes in lentiviruses, in which we demonstrate proof-of-concept for a 'template-switching' lentiviral vector that harnesses recombination events during reverse-transcription. During this work, we discovered that a standard, unmodified lentiviral vector was efficient in delivering full-length dystrophin to target cells, within a total genomic load of more than 15,000 base pairs. We have demonstrated gene therapy with this vector by restoring dystrophin expression in DMD myoblasts, where dystrophin was expressed at the sarcolemma of myotubes after myogenic differentiation. Ultimately, our work demonstrates proof-of-concept that lentiviruses can be used for permanent full-length dystrophin gene therapy, which presents a significant advancement in developing an effective treatment for DMD.

Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides.

Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.

Multifunctional, self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery.

Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5' RACE-PCR and Western blot analysis of protein.

The integration profile of EIAV-based vectors.

Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5' end of the transcription unit.