The Retroviridae family of viruses was first investigated as vectors for mammalian gene transfer over 30 years ago, and this technology continues to develop.
The HIV-1 elements that must be present in lentiviral vectors include the RNA packaging signal (Ψ), the major splice donor, and the Rev-response element (RRE), which are implicated in vector RNA processing and packaging into viral particles.
Following transduction of target cells, the RNA genome in a standard lentiviral vector is reverse-transcribed to form a double-stranded DNA (dsDNA) provirus. The HIV-1 long terminal repeats (LTRs) mark the boundaries of the reverse-transcribed template; thus, Ψ and RRE are always incorporated into standard lentiviral vector proviruses and integrated into the host cell genome as they are situated between the LTRs.
The persistence of HIV-1-derived elements in target cells presents a risk for clinical translation of lentiviral technology due to potential interactions between virus and patient genomes. The Ψ and RRE portions of lentiviral vector DNA contain functional cis elements, such as the major splice donor, which can disrupt cellular processes in patient cells. For example, it has been shown that integrated lentiviral proviruses can upregulate the human growth hormone receptor due to interactions between the HIV-1 major splice donor and splice acceptors in the human growth hormone receptor gene.
Previous attempts to remove Ψ and RRE from lentiviral vector proviruses have been largely unsuccessful. Cui et al.
Here, we describe the development and initial application of a novel lentiviral vector, LTR1, in which the HIV-1 packaging sequences have been relocated to avoid their transfer into target cell nuclei. In LTR1 vector RNA, the HIV-1 Ψ and RRE packaging sequences are located downstream of a single self-inactivating LTR (sin.LTR).
Optimization of LTR1 Genome to Achieve Vector Titers Sufficient for Gene Therapy
The premise of LTR1 technology is to generate an RNA genome that mimics the first strand that is synthesized during the initial stages of reverse-transcription in target cells (known as the “minus strand”). This is achieved in LTR1 vectors by removing the vector 5′LTR, thus leaving the HIV-1 primer binding site at the extreme 5′ terminus of the vector RNA. An additional primer binding site is situated immediately downstream of a solitary self-inactivating LTR (ΔU3 SIN), Figure S1, and the expected RNA and DNA products are displayed in Figure 1.
During iterative LTR1 development, vector transduction efficiency was determined by flow cytometry, by delivering GFP and the woodchuck-hepatitis virus post-transcriptional regulatory element (WPRE), driven by either the human phosphoglycerate kinase (PGK) promoter, or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The first iteration of LTR1 was produced by editing a third generation pRRL-PGK-EGFP-WPRE lentiviral plasmid (RRL-PEW), Figure 2A). This vector, termed LTR1.0-PGK-EGFP-WPRE (LTR.1.0-PEW), gave an infectious, EGFP titer of 1.2 × 105 transducing units per milliliter (TU/mL) following concentration by ultracentrifugation, almost three orders of magnitude lower than the standard RRL-PEW vector (3.4 × 108 TU/mL; data not shown), as determined by flow cytometric quantification of EGFP expression in transduced 293T cells (Figure 2B).
To increase LTR1 titers to a level suitable for gene therapy applications, the LTR1 genomic structure was progressively optimized with the goal of obtaining yields similar to the original third generation lentivirus from which LTR1 was developed (Figure 2A). Two major bottlenecks to producing high titer vector were believed to be insufficient nuclear export of vector RNA and undesirable transcription termination in the 3′sin.LTR. The majority of the optimization steps were designed to tackle these restrictions. The initial modifications that improved vector titers included exchanging the SV40 early polyA for the SV40 late polyA for improved polyadenylation (LTR1.5-PEW), use of the cytomegalovirus (CMV) promoter to increase expression of vector genomic RNA in producer cells (LTR1.7.671-GEW), and insertion of a small chimeric intron into the vector 5′ UTR with the intention of increasing nuclear export (LTR1.20-GEW).
Full-length LTR1 and CCL vector RNA would be expected to produce a 4 kb transcript, when all packaging elements are incorporated. However, northern blotting of vector RNA derived from LTR1.7.671 and LTR1.20 producer cells showed a strong band at approximately 1.7 kb when third generation packaging plasmids (without Tat expression) were used, while the wild-type CCL-GEW sample gave the expected 4 kb band at a greater frequency (Figure S2). Co-transfection with pcDNA3.Tat during vector production was able to rescue the 4 kb band in northern blotted LTR1.7.671-GEW and LTR1.20-GEW genomic RNA, indicating that efficient processing of LTR1 vector RNA during vector production is relatively dependent on the presence of HIV-1 Tat in producer cells. For this reason, Tat was included during the production of all LTR1 and CCL vectors for the remainder of the experiments.
The close proximity of the CMV enhancer to the internal GAPDH promoter in pLTR1.20-GEW and pLTR1.7.671-GEW may have strengthened the activity of this internal GAPDH promoter in producer cells, thus resulting in some vector transcripts being initiated from the GAPDH promoter itself (rather than CMV) and consequentially missing important upstream elements (primer binding site and cPPT). To combat potential CMV enhancement of the internal GAPDH promoter, we introduced larger introns in the 5′ UTR to effectively add space between the two promoters. Insertion of a truncated β-globin intron (LTR1.25-GEW) or an elongation factor 1 α intron (LTR1.27-GEW) was successful in increasing the functional titer (p = 0.004 by Kruskal-Wallis comparison of all vector titers) (Figure 2B). At this stage, LTR1.27 titers can be produced with efficiency equivalent to approximately 35% of a standard lentiviral vector (pCCL) titer.
LTR1 Proviruses Are Devoid of HIV-1 Packaging Sequences
The absence of HIV-1 packaging sequences from the delivered provirus is fundamental to the benefits of LTR1 technology. To confirm that the packaging signal is not copied during reverse-transcription, two methods were used to sequence LTR1-derived DNA proviruses in transduced cell lines.
Initially, a PCR was carried out using genomic DNA harvested from HT1080 cells transduced with an LTR1.20 vector containing EGFP expressed by the spleen focus-forming virus promoter (SFFV) (LTR1.20-SFFV-EGFP). The PCR was designed to amplify the region spanning from the 5′LTR-primer binding site junction up to the R component of the 3′LTR. Resolution of the PCR product by agarose gel electrophoresis showed that LTR1.20 products were approximately 2.4 kb, which matched the expected size of products lacking HIV-1 packaging sequences (Figure S3). This confirmed that LTR1.20-SFFV-EGFP products are indeed smaller than those derived from CCL-SFFV-EGFP, with an estimated provirus size difference of 1.3 kb. The sequence composition of the PCR products was confirmed by Sanger sequencing, which matched the expected provirus structures (data not shown).
A “plasmid rescue” method Figure S4). Sequencing of LTR1.20-rescue proviruses confirmed that the expected provirus structure, with Ψ and RRE removed, was present in transduced cells, and these products had successfully integrated into the cell genome with expected dinucleotide repeats (Figure S5).
LTR1 Transduction Characteristics Compared to Third Generation CCL In Vitro
In vitro experiments were carried out to further characterize LTR1 vectors and compare them to a third generation lentiviral vector. For these investigations, we used vectors containing an SFFV-driven bicistronic luciferase-EGFP reporter gene in which luciferase and EGFP are separated by a T2A cleavage peptide (SFFV-Luc-T2A-EGFP). Table 1).
Table 1Characterization of LTR1 and CCL Vector Composition by Various Titration Methods
|Vector||RNA Titer||Particle Titer||Provirus Titer||EGFP Titer||Reverse Transcription Efficiency (%)||Expression Efficiency (%)||Packaging Efficiency (%)|
|CCL||1.35 × 1011 ssRNAvg/mL||1.68 × 1011 lp/mL||2.23 × 1010 dsDNAvg/mL||1.62 × 1010 TU/mL||33.04||72.80||80.21|
|LTR1.20||2.24 × 1011 ssRNAvg/mL||2.60 × 1011 lp/mL||1.84 × 108 dsDNAvg/mL||1.09 × 108 TU/mL||0.16||58.96||86.32|
ssRNAvg/mL, ssRNA vector genomes per mL; lp/mL, lentiviral particles per mL; dsDNAvg/mL, dsDNA vector genome proviruses yielded per mL; TU/mL, EGFP-forming TU/mL.
The data in Table 1 show that, while LTR1.20-GEW particle and RNA genome titers are similar to CCL, the provirus titer and EGFP titer appear to be restricted. We further analyzed these parameters by calculating the approximate efficiency of key steps in the viral life-cycle. The relative packaging efficiency was calculated by expressing the RNA titer as a percentage of the particle titer, while the transgene expression efficiency was based on the EGFP titer as a percentage of the provirus titer. The efficiency of LTR1 packaging (86.3%) and transgene expression (59.0%) was similar to CCL (80.2% and 72.8%). Reverse-transcription efficiency was calculated by expressing the provirus titer as a percentage of the RNA genome titer. This value showed that LTR1.20 (0.16%) was less efficient than CCL (33.04%) at converting its RNA genome into a stable provirus.
The similarities and differences between LTR1.20 and CCL vector parameters are shown in more detail in Figure 3. Plotting the integrated VCN versus the mean fluorescence intensity (MFI) of EGFP expression (Figure 3A) shows that transgene expression efficiency per LTR1.20 provirus matches the level derived from the CCL vector. However, when plotting the p24 and RNA genome doses versus the percentage of GFP-positive cells (Figures 3B and 3C), it becomes apparent that LTR1.20 requires a greater total particle number and RNA dose than CCL to achieve an equivalent effect, underlining the potential inefficiency during transduction.
LTR1 Proviruses Are Resistant to Remobilization in the Presence of HIV-1 Packaging Components Provided in trans
Despite self-inactivating lentiviruses lacking an active promoter in the 5′LTR,
To investigate this, we transduced HEK293T cells with a 2-fold dilution series of LTR1.20-SFFV-EGFP or CCL-SFFV-EGFP and maintained the populations in culture for 11 days. Genomic DNA was harvested from each transduced population for VCN quantification, and the remaining cells were plated out and transfected with lentiviral packaging plasmids to create a pool of mock-HIV-1-infected cells. Vector supernatants were subsequently harvested and purified prior to addition to HT1080 cells, which were analyzed at 10 days post-transduction to quantify any expression from functional vector particles with the ability to deliver stably integrated remobilized viral genomes (Figure 4).
LTR1.20 samples were completely devoid of any remobilized proviruses, revealed by the lack of EGFP expression at all VCN doses. Third generation lentivirus (CCL) samples were positive for EGFP expression, with remobilized titers calculated in the range of 5.9 × 102–1.5 × 104 TU/mL, where area under the curve (AUC) increased in correlation with the starting VCN (p = 0.0004 by t test) (Figure 4A).
Three example flow cytometry dot plots show clear EGFP-positive colonies produced by a CCL-derived vector (Figure S6) remobilized from a HEK293T population possessing a CCL VCN of 2.03 vector genomes per cell. LTR1.20 was not remobilized from HEK293T cells containing 3.75 genomes per cell, with plots indistinguishable from a non-transduced negative controls.
The Risk of Generating Vector Host-Fusion Transcripts Is Reduced with LTR1 Technology
Transcripts derived from lentiviral vector proviruses have been shown to fuse with neighboring gene transcripts in patient cells through splicing interactions between HIV-1 splice sites and the human genome.
HEK293T cells were transduced with CCL-GEW, LTR1.7.671-GEW, or LTR1.20-GEW at a range of doses and expanded for 14 days to deplete any unintegrated proviruses. VCNs were calculated by qPCR (3.43 for CCL, 2.82 for LTR1.7.671, and 3.62 for LTR1.20). Total RNA was extracted from transduced cells and ribosomal RNA was depleted prior to preparation of next-generation sequencing libraries. Sequencing reads were analyzed for fusion transcripts by mapping paired reads to the human genome and subsequently to the relevant vector provirus, to find the frequency of human-vector transcript chimera per human genomic transcript. The number of fusion transcripts per 106 human transcripts was then normalized to the dsDNA provirus copy number in each sample.
The fusion transcript frequency detected in CCL samples (0.54 ± 0.06) was significantly greater than LTR1.7.671 samples (0.07 ± 0.02) and LTR1.20 samples (0.14 ± 0.02) (p = 0.027 by Kruskal-Wallis test) (Figure 4B). These values were also expressed relative to the number of CCL fusion transcripts, to highlight the reduced frequency of splice fusions when using LTR1.7.671 (12.97 ± 5.64% of CCL level) and LTR1.20 (26.5 ± 0.86% of CCL level) (p = 0.024 by Kruskal-Wallis test) (Figure 4C).
LTR1 Vectors Can Exceed Standard Lentiviral Vector Efficacy in the Liver following Murine Neonatal Intravenous Injection of Titer-Matched Viruses
To confirm that expression from an LTR1 provirus was detectable in vivo, LTR1.20-SFFV-EGFP (titer 2.34 × 107 TU/mL) was produced and administered intracranially or intravenously to newborn CD1 mice (Figure 5). Examination of EGFP expression 1 week after vector administration demonstrated that LTR1 could efficiently deliver transgene expression to mouse liver and brain (Figure 5A). Immunostaining of dissected brains demonstrated that stable EGFP expression existed predominantly within the cortex and hippocampus of the injected hemisphere (Figure 5B).
To compare longitudinal LTR1-driven in vivo expression to a CCL vector, the SFFV-Luc-T2A-EGFP bicistronic construct was used to allow bioluminescence imaging. Equivalent volumes of lentiviral vectors (LTR1.20 titer of 2.0 × 107 TU/mL and CCL titer of 3.6 × 107 TU/mL) were administered either intracranially or intravenously to newborn CD1 mice, which were then monitored for 36 days.
In vivo LTR1.20-SFFV-Luc-T2A-EGFP bioluminescence imaging revealed that the brain expression profiles of LTR1.20 did not show a statistically significant difference to CCL vectors (p = 0.087) (Figure 5C). However, comparison of luciferase expression in the livers of intravenously injected animals showed a significantly higher bioluminescent output from LTR1 vectors over the 36 day period (p = 0.018) (Figure 5D). The biodistribution and intensity of luciferase expression is shown in representative images at 0, 5, 15, and 36 days post-administration in Figure S7.
The Onset of Transgene Expression from LTR1 Vectors Occurs Earlier than Third Generation Lentiviral Vectors
An interesting observation was made when imaging the SFFV-Luc-T2A-EGFP animals immediately after vector administration. At just 20 min post-injection, luciferase expression was detectable in all LTR1.20-treated animals, but not in animals injected with third generation CCL vectors (Figure 6A).
In light of this finding, we sought to profile the timing of LTR1-derived expression during the initial stages after transduction. HT1080 cells were transduced at a MOI of 1 with LTR1.11.1-GEW, LTR1.13.0-GEW, or CCL-GEW (vector schematics can be found in Figure 2A). Flow cytometric analysis during the initial 48 hr after transduction revealed that the percentage of EGFP-positive cells derived from the LTR1.13.0 backbone was already at 8% just 1 hr after transduction, increasing at each subsequent time point (Figure 6B). Expression from the LTR1.11.1 and CCL backbones, which each contain a 5′LTR in their leader sequence, was minimal until 8 hr after transduction and increased thereafter. The percentage of EGFP-positive cells produced by LTR1.13.0 was significantly higher than LTR1.11.1 and CCL over the duration of the experiment, as determined by the differences between AUCs (p = 0.039 by Kruskal-Wallis multiple comparison test).
We looked deeper into the rapid onset of LTR1 expression by examining the timing of intracellular reverse-transcription products using late-RT qPCR Figure 6C). This analysis revealed that LTR1.13.0 reverse-transcription products were significantly greater than CCL over the initial 6 hr post-transduction (p = 0.036 by t test). LTR1.13.0 late-RT products were detectable by 1 hr post-injection, at a level not matched by CCL until 4 hr post-injection (Figure 6C). LTR1.11.1 did not produce late-RT products significantly faster than CCL (p = 0.057), suggesting that minus-strand transfer may be an influential factor in the onset of LTR1 expression.
LTR1 Vectors Can Be Used to Correct a Factor IX-Deficient Mouse Model
To demonstrate gene therapy with LTR1 technology, we sought to correct a factor IX (FIX)-deficient mouse model of hemophilia B by in vivo FIX gene transfer. For this, we used codon-optimized factor IX cDNA (FIX) containing the hyperactive Padua mutation.
Vectors were delivered to neonatal mice by intravenous administration at post-natal day 1. The vector doses were determined by the total number of administered viral genomes, with doses being 1.4 × 108 dsDNAvg for CCL, 1.7 × 107 dsDNAvg for LTR1.25, and 1.5 × 108 dsDNAvg for LTR1.27. Mouse livers and plasma samples were collected upon termination of the experiment. The liver proviral copy number was determined by qPCR, which were calculated as being 1.8 ± 0.9 for LTR1.25, 1.7 ± 0.9 for LTR1.27, and 1.4 ± 0.5 for CCL (Figure 7A).
LTR1.25-treated mouse plasma contained mean factor IX protein levels of 12.2 ± 5.2% of normal reference levels, which was similar to the 12.3 ± 3.7% produced by CCL (Figure 7B). Mean plasma factor IX activity was raised to 14.9 ± 7.5% normal levels by LTR1.25, which again matched the CCL mean output of 12.6 ± 4.0% (Figure 7C). LTR1.27 treatment gave 22.9 ± 1.7% factor IX protein levels and 24.4 ± 3.0% factor IX activity. No statistically significant difference was detected during analysis of plasma factor IX protein (p = 0.153 by Kruskal-Wallis) and activity (p = 0.35 by Kruskal-Wallis). The overall level of factor IX restoration would be sufficient for corrective gene therapy in humans,
Lentiviruses offer a number of advantages as gene therapy vectors and their development continues to progress.
We have developed a lentiviral vector with a novel formation that ensures minimal transfer of wild-type HIV-1 sequences. The LTR1 reverse-transcription mechanism is unprecedented in retrovirology, as all other retroviruses and retrovirus-derived vectors require at least two strand transfer events to complete proviral synthesis. In LTR1 vectors, minus-strand synthesis is primed adjacent to a solitary 3′LTR, thus rendering the usual first strand transfer event obsolete. This demonstrates that the first strand transfer event in reverse-transcription is not necessary for functional lentiviral transduction of target cells, revealing an interesting facet of HIV biology. Importantly, this potentially constitutes a shortening of the viral life-cycle and increased in vivo efficacy, analogous to self-complementary AAV (scAAV).
The structure of the LTR1 provirus reveals interesting insights into HIV biology and brings an important technological advancement in terms of the potential safety of clinical gene therapy. HIV-1 RNA packaging sequences comprise approximately 1.9 kb of its genome, meaning that third generation lentiviruses bear 20% sequence homology with wild-type HIV-1. Here, and in previous studies, lentiviral proviruses have been shown to produce full-length RNA genomes that can be remobilized in replicating HIV-1 particles, even with the use of a self-inactivating LTR.
HIV-1 packaging sequences contain active splice donor and splice acceptor sites, which have previously proven very difficult to remove from vector genomes.
We have shown that LTR1 vectors can be produced at titers sufficient for pre-clinical gene therapy. We demonstrated LTR1-mediated correction in a hemophilia B mouse model, in which LTR1.25 and LTR1.27 were able to deliver liver VCNs equal to a standard CCL lentiviral vector. Plasma factor IX expression was detected at therapeutic levels and with sufficient biological activity, upon termination of the experiment, indicating that LTR1 could be used for stable disease correction in a gene therapy setting.
Close analysis showed that LTR1.27 restored mouse plasma factor IX activity to around 24% of normal levels when delivered at a similar dose to CCL, which returned around 13%. The level of factor IX expression derived per injected LTR1.27 genome is an intriguing matter for further investigation. While encouraging, it cannot be ignored that all vector doses were based on the infectious titer, which we have identified as being lower than the physical particle titer in the case of LTR1.20, compared to CCL. Therefore, there is a possibility that excess defective particles were also injected along with functional LTR1.25 and LTR1.27 particles, potentially impacting on transduction. Possible mechanisms might include blocking clearance of particles in cells such as Kupffer cells, thus allowing more vector particles to infiltrate hepatocytes.
During longitudinal tracking of luciferase expression in neonatally injected CD1 mice, LTR1 showed significantly more liver bioluminescence than a titer-matched third generation CCL over the course of the experiment (36 days). The exact mechanism for this difference is unclear, and the influence of excess defective particles cannot be ruled out, considering that the CCL profile appears to match LTR1 at early stages of the investigation before decreasing from day 18, which was less pronounced with LTR1. This profile was not observed following brain transduction.
An intriguing feature witnessed during LTR1 characterization was the rapid onset of transgene expression. This was observed both in vitro and in vivo, with two separate vector transgene cassettes. Following intracerebral and intravenous LTR1.20-luciferase injections, mice were immediately examined for bioluminescence. This investigation revealed that LTR1.20 was capable of producing luciferase expression 20 min after injection, potentially indicating a unique functionality that could be exploited. This early level of expression was not provided by third generation CCL. Leading on from this finding, we sought to investigate how the structure of LTR1 RNA could influence the timing of transgene expression in vitro. This experiment revealed that when LTR1 was lacking a 5′LTR (LTR1.13.0; Figure 2A), there was a significant acceleration of transgene expression during the initial 48 hr after transduction. Again, this may relate to the scAAV vector paradigm in which shortening of the replication phase of transduction improves the speed and efficiency of transgene expression.
The data concerning the onset of reverse-transcription products suggest that the smaller LTR1 genome and unique reverse-transcription pathway may facilitate rapid copying of its RNA genome, thus accelerating the onset of gene expression. Interestingly, LTR1.11.1, which does perform both strand transfer events, also produced reverse-transcription products earlier than a standard vector, suggesting that minus-strand transfer is not the exclusive reason for differences in the speed of transduction. The size of the LTR1.11.1 provirus is approximately 1.5 kb smaller than that of the standard CCL vector, thus the speed of transduction may be partly influenced by the size and complexity of the template to be copied. This rapid onset of expression constitutes a unique feature of LTR1 that may be exploited for gene therapy purposes in scenarios requiring vector expression within a short time frame. This could be particularly advantageous during ex vivo manipulation of stem cells and when using non-integrating lentiviral vectors, given that unintegrated proviruses are rapidly lost from dividing cells after repeated passages.
Our extensive characterization of LTR1 vectors has revealed interesting features that will be important to consider when moving forward with further LTR1 development. During northern blot analysis of packaged vector genomes, we discovered that LTR1 vectors are dependent on the expression of HIV-1 Tat in producer cells for efficient transcription of full-length viral genomes. We observed that shorter transcripts were generated in the absence of Tat, presumably due to undesirable termination of transcription and poly-adenylation at the solitary LTR. We expect that this was rescued by Tat due to its ability to promote transcriptional readthrough of the 3′LTR through its binding the HIV-1 trans-activation response element (TAR).
Gene therapy with lentiviral vectors is being pursued for a rapidly increasing cadre of diseases including beta globinopathies, chronic granulomatous disease, leukodystrophies, and blood cancers.
Materials and Methods
Generation of Plasmid Constructs
All plasmid constructs were made using standard molecular cloning procedures and PCR-mediated deletion of plasmid sequences.
Cell Culture Maintenance
HEK293T cells were used for production of viral vectors, remobilization assays, and for analysis of fusion transcripts. HT1080, a human fibrosarcoma cell line, was used for titration of VCNs by qPCR. HEK293T and HT1080 cell lines were cultured at 37°C in DMEM (Invitrogen) supplemented with 10% (v/v) fetal bovine serum. All lines were split three times per week, when ∼80% confluence was reached.
Production of Lentiviral Vectors
Lentiviruses were produced using a second-generation packaging system as described previously.
Titration of Lentiviral Vectors
Vector titration by flow cytometry: 1 × 105 HEK293T cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, cells were trypsinized and EGFP-positive cells were quantified using a BD Cyan flow cytometer or BD FACSArray Bioanalyzer 3 days after transduction.
HEK293T cells were not used for qPCR titration due to their reported abnormal karyotype.
Detection of EGFP Expression in Transduced Cells
Flow cytometric detection of EGFP expression was used for titration and characterization of LTR1 vectors. Unless stated otherwise, 100,000 cells were analyzed for detection EGFP expression in a BD FACSArray Bioanalyzer. EGFP fluorescence was excited using a 488 nm laser. During analysis of flow cytometry plots, cells were gated by plotting forward-light-scatter versus side-scatter to isolate the live population. EGFP-positive populations were identified by plotting EGFP fluorescence (detected using a 530/30 nm band-pass filter) versus emission from the yellow channel (detected using a 575/26 band-pass filter) to compensate for auto-fluorescent events. Non-transduced cell populations were used as negative controls to set the background level of emission in each channel. All flow cytometry data were analyzed by FlowJo software version 9.3.1 (Tree Star).
Dose-Response Profiling of LTR1 Vectors versus Third Generation Lentiviral Vectors In Vitro
HT1080 cells were infected with either LTR1.20-SFFV-Luc-T2A-EGFP or CCL-SFFV-Luc-T2A-EGFP at a range of MOI, prepared by serial 2-fold dilutions of starting stocks. To avoid the influence of unintegrated lentiviral expression, cells were expanded for 14 days before analysis by flow cytometry and genomic DNA extraction for proviral copy number analysis. The starting vector stock was assayed for p24 concentration by p24 ELISA and the vector RNA genome titer was calculated by qRT-PCR, with methods described in Titration of Lentiviral Vectors.
PCR Analysis of Integrated LTR1 Proviruses
HT1080 cells were transduced with either LTR1.20-SFFV-EGFP or CCL-SFFV-EGFP at an MOI of 10. At 2 weeks after transduction, cells were harvested and genomic DNA was extracted using a DNeasy Blood and Tissue kit (QIAGEN). To determine the size of the vector backbone, genomic DNA was PCR-amplified using oligos specific for the lentiviral 5′LTR-primer binding site junction (5′-AAATCTCTAGCAGTGGCGCCCGAACAG-3′) and the 3′LTR R region (5′-GCACTCAAGGCAAGCTTTATTGAGGCTT-3′).
Northern Blotting of Vector RNA
RNA species from pCCL, pLTR.1.7.671, and pLTR1.20 were analyzed during particle production or after proviral integration. For analyzing vector genomes during packaging, 107 HEK293T cells were transfected with 12 μg pcDNA3.HIV-1g/p.4×CTE,
Examination of Provirus Structure by Plasmid Rescue
The CCL-Rescue and LTR1.20-Rescue plasmids were produced by excising the pBR322 Figure S4). HEK293T cells were plated in 6-well plates at a density of 1 × 105 cells per well and transduced with either 10 μL (0.1 μg p24) of concentrated CCL-Rescue or 200 μL (15 μg p24) of concentrated LTR1.20-Rescue. Cells were maintained in culture for 2 weeks before extracting genomic DNA. For each sample, 10 μg of genomic DNA was treated with XbaI (to ensure extensive cutting of the human genome while avoiding digestion of viral genomes) for 1 hr before column purification (QIAGEN PCR Purification Kit - 28104) and ligation. Electrocompetent Stbl4 cells (Life Technologies) were transformed with the ligated sample in a 0.1 mm electroporation cuvette at a frequency of 1.2 kHz and 25 μF capacitance. Transformed bacteria were then selected on agar plates (100 μg/mL ampicillin) to isolate any provirus-containing colonies, from which plasmid DNA was harvested and screened for the presence of lentiviral proviruses by targeted restriction digest of lentiviral LTRs (with AflII) extracted plasmid DNA. The provirus-containing plasmids were subsequently sequenced to determine the composition of integrated LTR1.20 and CCL proviruses.
Detection of Remobilized Self-Inactivating Lentiviral Genomes
HEK293T cells were transduced with CCL-SFFV-EGFP or LTR1.20-SFFV-EGFP with a range of vector doses. At 11 days after transduction, a sample of each population was taken for genomic DNA extraction and qPCR quantification of VCNs. For each production replicate, 1.8 × 107 transduced cells were seeded into T175 flasks. At 24 hr later, each flask was transfected with 30 μg of pCMVΔR8.74 and 10 μg of pMDG.2. DNA was mixed in 5 mL Opti-MEM and filtered through a 0.22 μm filter before combining with 5 mL Opti-MEM containing 1 μL PEI (10 mM). The resulting 10 mL mixture was applied to the transduced cells after 20 min incubation at room temperature. The transfection mix was removed after 4 hr and replaced with fresh culture medium. Lentiviral supernatants were processed as described in Production of Lentiviral Vectors. Fresh HEK293T cells were seeded in 6-well plates at a density of 1 × 105 cells per well and transduced with 50 μL of the concentrated viruses (n = 3). At 11 days after transduction, cells were analyzed by flow cytometry to detect the number of EGFP-expressing cells. Cells were analyzed in a BD FACSArray Bioanalyzer and non-transduced cells provided the baseline for background fluorescence.
Transcriptomic Profiling of Vector-Host Fusion Transcripts
HEK293T cells were transduced with CCL-GAPDH-EGFP, LTR1.7.671-GAPDH-EGFP, or LTR1.20-GAPDH-EGFP at a range of doses and VCNs quantified by qPCR. Samples with similarly matched copy numbers were processed for transcriptomic profiling. Total RNA was extracted from cells and 1 μg was processed, with ribosomal RNA depleted using the Kapa RiboErase kit (Kapa Biosystems - KK8483). RNA was fragmented to produce intact fragments of 200–300 bp and adaptor-ligated sequencing libraries were prepared according to the manufacturer’s protocol. Libraries were sequenced on the Illumina NextSeq system. FASTQ files were analyzed on the Galaxy platform http://www.usegalaxy.org instance) by mapping paired reads to the human genome (hg38) using HISAT
For in vivo investigations, outbred CD1 mice (Charles River), or hemophilia B mice Figure 6A. All experiments were performed in accordance with relevant guidelines and regulations. Experiments were carried out under United Kingdom Home Office regulations and approved by the ethical review committee of University College London.
Confirmation of Vector Efficacy In Vivo by Luciferase Expression
To monitor LTR1 bioluminescence in vivo, LTR1.20-SFFV-Luc-T2A-EGFP (2.0 × 107 TU/mL) or CCL-SFFV-Luc-T2A-EGFP vectors (3.6 × 107 TU/mL) were administered either intracranially (2 × 5 μL bilaterally) or intravenously (40 μL) to 1-day-old neonatal CD-1 mice. Images and bioluminescence data were gathered continually for 36 days as described previously.
Immunohistochemistry Staining and EGFP Imaging of LTR1.20-SFFV-EGFP Transduced Brains
CD-1 mice were injected with LTR1.20-SFFV-GFP (2.34 × 107 TU/mL) at post-natal day 1. Mice received vector either by direct intracranial injection into the left lateral ventricle (5 μL), or intravenously (20 μL). At 1 week later, they were sacrificed and organs imaged by fluorescence microscopy (Leica MZ16) or immunohistochemistry. For immunohistochemistry, brains were embedded in paraffin wax and sliced in the coronal plane in preparation for EGFP-staining. Brain sections were treated with 30% H2O2 in tris-buffered saline (TBS) for 30 min and blocked with 15% of goat serum (Vector Laboratories) in TBS-tween 20 (TBST) for 30 min. Rabbit anti-GFP (1:10,000; Abcam) was added in 10% goat serum in TBST and left on a gentle shaker overnight at 4°C. Goat anti-rabbit (1:1,000; Vector Laboratories) was then added in 10% goat serum in TBST for 2 hr. The sections were incubated for a further 2 hr with VECTASTAIN ABC (Vector Laboratories), followed by addition of 0.05% 3,3′-diaminobenzidine (DAB) and brief incubation. Sections were transferred to ice-cold TBS. Each individual brain section was mounted on chrome gelatin-coated Superfrost Plus slides (VWR) and left to dry for 24 hr. The slides were dehydrated in 100% ethanol and placed in Histo-Clear (National Diagnostics) for 5 min before mounting with DPX mounting medium (VWR).
Monitoring In Vitro EGFP Expression during Early Stages of LTR1 Transduction
HT1080 cells were plated onto 6-well plates at a density of 1 × 105 cells per well in a 1 mL volume and simultaneously transduced with LTR1.11.1-GAPDH-EGFP, LTR1.13.0-GAPDH-EGFP, or CCL-GAPDH-EGFP at an MOI of 1. At various time points after transduction, cells were trypsinzed and harvested for flow cytometric analysis of EGFP expression as described in Titration of Lentiviral Vectors. Genomic DNA was extracted from cell pellets and analyzed for reverse-transcription products using a previously reported late-RT qPCR assay.
LTR1-Mediated FIX Delivery
A codon-optimized version of FIX containing the hyperactive Padua mutation
All statistical analyses were carried out using either MATLAB 2015a or Python SciPy open-source software.
Data and Materials Availability
C.A.V. and S.J.H. - design of initial concept, execution and analysis of experiments, and review of paper; J.R.C. - design, execution and analysis of experiments, and writing paper; and S.N.W., T.R.M., R.K., D.P.P., S.M.K.B., M.H.B., A.S., and M.G. - design, execution and analysis of experiments, and review of paper.
Conflicts of Interest
C.A.V. and S.J.H. are named inventors on LTR1 technology international patent (patent application number PCT/GB2014/053102). All other authors declare no competing interests.
We are grateful for the help provided by Mr. Ayad Eddaouadi at the Institute of Child Health FACS facility for his technical assistance and advice during the gathering and interpretation of flow cytometry data. We thank Dr. Tony Brooks and Dr. Mike Hubank of UCL Genomics for their assistance and advice in executing next generation sequencing experiments. We also thank Thomas Neumann at Hannover Medical School for his technical assistance in performing northern blots of vector genomic RNA. We are also grateful to Dr. Luis Apolonia of King’s College London for reviewing the accuracy and reporting of HIV-1-relevant material. J.R.C. was funded by a BBSRC Follow-On Fund and MRC Confidence in Concept grant . A.S. and M.G. were supported by grants from the German Research Council ( DFG/SFB738 and DFG/ REBIRTH ). T.R.M. and S.N.W. were funded by NC3Rs grant NC/L001780/1 . Initial work was supported by a BBSRC New Investigator Award BB/I00212X/1 (to S.J.H. and C.A.V.).
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Published online: May 24, 2017
Accepted: April 28, 2017
Received: December 25, 2016
© 2017 The Author(s).
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Figure 1The Structure of LTR1 Gives Rise to RNA and DNA Products that Are Distinct from CCL
Figure 2Key Stages of the LTR1 Development Process
Figure 3Characterization of LTR1 Vector Titers In Vitro
Figure 4Analyzing the Safety Parameters of CCL and LTR1 Vector Proviruses
Figure 5LTR1 Vectors Can Be Used for Efficient In Vivo Gene Delivery
Figure 6LTR1 Vectors Produce Transgene Expression Earlier than Standard Lentiviruses
Figure 7LTR1 Increases Plasma Factor IX Expression following Treatment of a FIX-Deficient Mouse Model