Lentiviral Vector Titers Research Articles

Genetic alteration of SJ293TS cells and modification of serum-free media enhances lentiviral vector production

Successful cell and gene therapy clinical trials have resulted in the United States Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies, which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells, have driven additional investigations, increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods, we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media, SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold, while absence of Beta-2 microglobulin, a key component of Major Histocompatibility Complex class I molecules, has been reported to reduce the immunogenicity of lentiviral particles. Furthermore, we describe an improved methodology for culturing SJ293TS-DPB which facilitates expansion, reduces handling, and increases titers by 2-fold compared to previous methods. SJ293TS-DPB stably produced lentiviral vectors for over four months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34+ hematopoietic stem cells.

CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers.

Lentiviral (LV) vectors have emerged as powerful tools for treating genetic and acquired human diseases. As clinical studies and commercial demands have progressed, there has been a growing need for large amounts of purified LV vectors. To help meet this demand, we developed CRISPR library screening methods to identify genetic perturbations in human embryonic kidney 293 (HEK293) cells and their derivatives that may increase LV vector titers. Briefly, LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) were used to modify HEK293 and HEK293T cells. These cell populations were then expanded, and integrated LV vector genomes were rescued by transfection. LV vectors were harvested, and the process of sequential transduction and rescue-transfection was iterated. Through this workflow, guide RNAs (gRNAs) that target genes that may suppress or enhance LV vector production were enriched and identified with Next-Generation Sequencing (NGS). Though more work is needed to test genes identified in this screen, we expect that perturbations of genes we identified here, such as TTLL12, which is an inhibitor of antiviral innate immunity may be introduced and multiplexed to yield cell lines with improved LV vector productivity.

A truncated reverse transcriptase enhances prime editing by split AAV vectors

Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for invivo applications.

Improved lentiviral vector titers from a multi-gene knockout packaging line

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2'-5'-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

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.

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

Promoter’s Choice Drive CAR-T Efficiency

The development and the functionality of Chimeric Antigen Receptor (CAR) therapy is highly linked to the stability and expression level of the receptor, for which the choice of the promoter is key. Although EF1α is a reference promoter to induce strong antigen binding and long-term CAR expression, we investigated the impact of this one and other constitutive promoters (MSCV, PGK, β2m and CMV) on a CAR123 construct in the Blastic Plasmocytoid Dendritic Cell Neoplasm (BPDCN). Our results indicate that lentiviral vector titer is affected by transgene nature and promoter activity. Interestingly, by moderating the expression level of CAR123 at the cell surface, its recognition capacity and selectivity were improved. We observed that in early stages after transduction, CAR123 expression level did not affect lymphocyte’s phenotype and its cytotoxic capacity against BPDCN cell line. However, following long-term culture and decrease of cell-surface CAR expression, a hallmark of transition from activation to memory state of T-cells was observed, and only T-cells transduced with CAR-EF1α or -β2m promoters were still functional. We identified β2m as a promising promoter. Though it shows lower antigen-binding CAR-T cell and antigen recognition rates compared to EF1α, β2m promoter still present interesting CAR expression levels, stability, and binding capacity

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA.

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60-80% to 30-40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.

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.

Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity

Most gene therapy lentiviral vector (LV) production platforms employ HEK293T cells expressing the oncogenic SV40 large T-antigen (TAg) that is thought to promote plasmid-mediated gene expression. Studies on other viral oncogenes suggest that TAg may also inhibit the intracellular autonomous innate immune system that triggers defensive antiviral responses upon detection of viral components by cytosolic sensors. Here we show that an innate response can be generated after HIV-1-derived LV transfection in HEK293T cells, particularly by the transgene, yet, remarkably, this had no effect on LV titer. Further, overexpression of DNA sensing pathway components led to expression of inflammatory cytokine and interferon (IFN) stimulated genes but did not result in detectable IFN or CXCL10 and had no impact on LV titer. Exogenous IFN-β also did not affect LV production or transduction efficiency in primary T cells. Additionally, manipulation of TAg did not affect innate antiviral responses, but stable expression of TAg boosted vector production in HEK293 cells. Our findings demonstrate a measure of innate immune competence in HEK293T cells but, crucially, show that activation of inflammatory signaling is uncoupled from cytokine secretion in these cells. This provides new mechanistic insight into the unique suitability of HEK293T cells for LV manufacture.

Transient Lentiviral Vector Production Using a Packed-Bed Bioreactor System.

Scalable lentiviral vector (LV) manufacturing is vital for successful commercialization of LV-based gene and cell therapy products. Accordingly, efforts are currently focused on developing and adapting technologies to address both upstream and downstream production bottlenecks. To overcome the limitations of current upstream processes, researchers are now favoring the use of bioreactors over traditional two-dimensional culture platforms. Bioreactors provide many advantages for manufacturing biomolecules, including process automation, tight regulation of production conditions, reduced labor input, and higher productivity potential. This study describes a transient LV production strategy employing a single-use, packed-bed bioreactor vessel. Functional LV titers in the 106 TU/mL range were achieved, and after concentration yields of up to 109 TU/mL were attained. This proof of principle study demonstrates that LV can be successfully produced in a packed-bed system. With further optimization, a packed-bed bioreactor could offer a potential scale-out solution for LV manufacturing.

Improvement ofDe NovoCholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors

The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.

Regulation of Postoperative Ileus by Lentivirus-Mediated HuR RNA Interference via the p38/MK2 Signaling Pathway

ObjectivesThe objective of the present study is to explore the effect of lentivirus-mediated HuR interference on the development and progression of postoperative ileus and the role of HuR in the regulation of the p38/MAPK-activated protein kinase-2 (MK2) signaling pathway during postoperative ileus. MethodsTo establish a mouse model of lentiviral transduction, we first determined the optimum effective titer of lentiviral vectors for transduction of the murine small intestine via the abdominal cavity by using hematoxylin and eosin (HE) staining, immunohistochemistry, detection of GFP messenger RNA (mRNA) and protein, and Western blotting. To investigate the effect of HuR interference on gene expression during postoperative ileus, we established a mouse model of postoperative ileus and used RT-PCR to measure the expression of proinflammatory genes, ELISA to measure the expression of serum inflammatory cytokines, immunohistochemistry to evaluate inflammatory cell infiltration in the small intestine, HE staining of paraffin sections to examine the pathology of the small intestine, and Western blotting to measure HuR expression and identify its role in the regulation of the p38/MK2 inflammatory pathway. ResultsWe successfully designed a mouse model of intraperitoneal transduction of HuR-RNAi lentivirus. When HuR gene expression was suppressed in a mouse model of postoperative ileus, the infiltration of inflammatory cells, the expression of proinflammatory genes, and the levels of serum inflammatory cytokines were significantly reduced. This reduction in inflammation correlated with reduced cytoplasmic localization of HuR and reduced activation of MK2. ConclusionsWithin the p38/MK2 signal transduction pathway, HuR may increase the mRNA stability of various inflammatory cytokines, thereby promoting inflammation that causes postoperative ileus. Suppressing the expression of HuR in a postoperative ileus model can effectively suppress the postoperative ileus inflammatory reaction. HuR might serve as a candidate drug target for the prevention and mitigation of postoperative ileus.

Optimizing Transgene Configuration and Protein Fusions to Maximize Dopamine Production for the Gene Therapy of Parkinson's Disease.

Pharmacological dopamine replacement therapies provide the most well-established treatments for Parkinson's disease (PD). However, these long-term treatments can lead to motor complications and off-target effects. ProSavin(®), a lentiviral vector (LV)-based gene therapy approach aimed at restoring local and continuous dopamine production, through delivery of three enzymes in the dopamine biosynthesis pathway, was demonstrated to be safe and well-tolerated in a phase I/II clinical study of patients with advanced PD. Although improvements in motor behaviour were observed, the data indicated that higher levels of dopamine replacement might be required to maximize benefit. We attempted to increase production of dopamine, and its precursor L-Dopa in LV-transduced cells, by optimizing the gene order in the ProSavin expression cassette, and by creating fusions of two or three of the transgenes, using linker sequences. In vitro analysis showed that several gene arrangements provided significantly increased dopamine and/or L-Dopa production compared with ProSavin, and that LV titers and transgene expression were not affected by introducing gene fusions. One vector, equine infectious anemia virus (EIAV)-TCiA, was selected for further characterization and showed significant improvements in dopamine and L-Dopa production compared with ProSavin, in human neuronal cells. Further characterization of EIAV-TCiA demonstrated expression of all three dopamine enzymes in vivo and faithful delivery and integration of the expected gene expression cassette within the genome of target cells, as assessed by Northern and Southern blotting. In conclusion, we have developed a novel LV vector with an increased capacity for L-Dopa and dopamine production compared with the current ProSavin vector. Clinical evaluation of this vector will be performed to assess the benefits in patients with PD.

Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production.

BackgroundHigh titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector.MethodsWe prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K+ channels, using the ubiquitously active β-actin and neuron-specific synapsin I promoters.ResultsThe titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K+ currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba2+ during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K+ concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba2+ increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K+ currents generated by the channels expressed.ConclusionIn the production of lentivirus expressing K+ channels, titers are increased by blocking K+ currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

Universal Real-Time PCR-Based Assay for Lentiviral Titration

Lentiviral vectors are efficient vehicles for stable gene transfer in both dividing and non-dividing cells. This feature among others makes lentiviral vectors a powerful tool in molecular research. However, the use of lentiviruses in research studies and clinical trials requires a precise and validated titration method. In this study, we describe a qPCR-based approach for estimation of lentiviral vector titer (pLV-THM-GFP). The use of WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) and albumin genes as templates for an SYBR green-based real-time qPCR method allows for a rapid, sensitive, reproducible, and accurate assessment of lentiviral copy number at an integrated lentiviral DNA level. Furthermore, this optimization enables measurement of lentiviral concentration even in very poor quality and small quantity material. Consequently, this approach provides researchers with a tool to perform low-cost assessment with highly repeatable results.

Influence of mildly acidic pH conditions on the production of lentiviral and retroviral vectors.

Human immunodeficiency virus type 1-derived lentiviral vectors (LVs) are becoming major tools for gene transfer approaches. Several gene therapy clinical studies involving LVs are currently ongoing. Industrial production of clinical-grade LVs is therefore an important challenge. Some improvements in LV production protocols have already been possible by acting on multiple steps of the production process like transfection, cell culture, or media optimizations. Yet, the effects of physicochemical parameters such as pH remain poorly studied. Mammalian cell cultures are generally performed at neutral pH, which may not be the optimal condition to produce high quantities of LVs with optimal infectious properties. In this study, we showed that lentiviral transient production in HEK293T cells is inversely dependent on the pH value of the harvesting medium. Infectious and physical titers of LVs pseudotyped with GALVTR or VSV-G glycoproteins are enhanced by two- to threefold at pH 6 compared with neutral conditions. pH 6-produced LVs are highly infectious on cell lines but also on relevant primary target cells like hCD34+ hematopoietic stem/progenitor cells. GALVTR-LV particles produced at pH 6 are highly stable at 37 °C and resistant to multiple freeze-thaw cycles. Higher levels of expression of intracellular pr55gag polyproteins are observed within HEK293T producer cells cultured at pH 6. The positive effect of pH 6 conditions is also observed for moloney-derived retroviral vectors produced from NIH-3T3 fibroblasts, arguing that the mildly acidic pH effect is not limited to the lentivirus genus and is reproducible in various producer cell lines. This observation may help us in the design of more effective LV production protocols for clinical applications.

The HIV-1 Rev Protein Enhances Encapsidation of Unspliced and Spliced, RRE-Containing Lentiviral Vector RNA

BackgroundDuring the RNA encapsidation process of human immunodeficiency virus (HIV) viral genomic, unspliced RNA (gRNA) is preferentially incorporated into assembling virions. However, a certain amount of spliced viral transcripts can also be detected in viral particles. Recently, we observed that nuclear export of HIV and lentiviral vector gRNA by Rev is required for efficient encapsidation. Since singly-spliced HIV transcripts also contain the Rev-response element (RRE), we investigated if the encapsidation efficiency of RRE-containing spliced HIV-vector transcripts is also increased by the viral Rev protein.FindingsStarting with a lentiviral vector imitating the splicing pattern of HIV, we constructed vectors that express an unspliced transcript either identical in sequence to the singly-spliced or the fully-spliced RNA of the parental construct. After transfection of the different lentiviral vectors cytoplasmic and virion-associated RNA levels and vector titers were determined in the presence and absence of Rev. Rev enhanced the infectious titer of vectors containing an RRE 6 to 37-fold. Furthermore, Rev strongly increased encapsidation efficiencies of all RRE-containing transcripts up to 200-fold. However, a good correlation between encapsidation efficiency and lentiviral vector titer could only be observed for the gRNA. The infectious titer of the vector encoding the fully-spliced RNA without RRE as well as the encapsidation efficiency of all transcripts lacking the RRE was not influenced by Rev. Interestingly, the splicing process itself did not seem to interfere with packaging, since the encapsidation efficiencies of the same RNA expressed either by splicing or as an unspliced transcript did not differ significantly.ConclusionsRev-mediated nuclear export enhances the encapsidation efficiency of RRE-containing lentiviral vector RNAs independently of whether they have been spliced or not.

Optimization of the Transductional Efficiency of Lentiviral Vectors: Effect of Sera and Polycations

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.