Robust Lentivirus Packaging Platform

A cell will serve as a target for lentivirus transduction if it has cell surface receptors of adequate density and affinity for the env protein being expressed by a given lentivirus. Often recombinant lentiviruses are engineered to express the heterologous VSV-G env protein on the surface, resulting in broad species and cell-type transduction. The choice of the ideal env to incorporate into a given lentivirus is dictated by the target cell type being studied.

The two key safety concerns when working with recombinant lentiviruses, and how they are avoided, are as follows:

1. The generation of replication-competent lentivirus (RCL) – From a cell culture process perspective, implementation of BSL2 laboratory safety practices is mandatory. From a vector design perspective utilizing 3rd generation (4-plasmid) methodologies minimize the likelihood of RCL generation. The lentiviral packaging components are separated across two plasmids (gag & pol on one plasmid, rev on a second plasmid). Additionally, the lentiviral tat gene is omitted from the system. Lastly, the lentiviral LTRs are commonly engineered with Self-Inactivating (SIN) modifications to further reduce the risk of RCL generation.


2. The potential for oncogenesis – The mechanism of lentivirus integration is not well understood. Genome-wide assessments of viral integration have established a pattern of preferential integration into actively transcribed genes. The likelihood for oncogenesis is associated with the nature of the gene-of-interest (GOI) being engineered into the transfer/shuttle plasmid. If the GOI has homology to an oncogene or tumor suppressor gene, care must be taken in the design of the vector to minimize oncogenic integration events (minimizing the chances of oncogene activation or tumor suppressor gene inactivation). Care must also be taken pre-clinically to assess the occurrence of high-risk integration events post-transduction.

Lentivirus stocks can be titered biophysically (# of particles/ml) or functionally (transduction units/ml) by transducing a relevant target cell with serially-diluted lentivirus stocks.

Particle titers are most often assessed utilizing a p24 ELISA. Multiple companies offer p24 ELISA kits and services. Other consistently expressed lentivirus proteins can also be the readout for a biophysical assay.

Functional titer assays can be carried out via cell culture-based transduction assays, by molecular RT-PCR assays, or by flow cytometry assays (if the lentivirus expresses a fluorescent reporter gene or a cell surface protein with commercially available antibodies).

Estimated infectious titer by qPCR is routinely utilized to give a best estimated infectious titer using qPCR for research-grade lentivirus titration. If an accurate infectious titer/transduction unit is needed, then a cell culture-based transduction assay is preferred.

Lentivirus are prone to losing viability (functional titer) upon cryopreservation and during repeated freeze/thaw (F/T) cycles. uBrigene has developed cryopreservation methods and reagents which lead to more robust lentiviral particles with enhanced –80°C and 4°C viability, resulting in greater experimental reproducibility.

This is driven by the experimental goal, and the target cell type. Lentiviruses have become a valuable vehicle for the overexpression or suppression of a genetic target.

Overexpression of a gene-of-interest (GOI) is driven by RNA Pol II. An overexpression cassette should contain a Pol II-compatible enhancer/promoter. Heterologous viral regulatory elements are typically utilized (often CMV or RSV). Cell-specific enhancer/promoter cassettes can also be implemented when cell-specific expression is an important experimental goal.

The most common technology used for suppression of gene expression is shRNA. Expression of shRNA is controlled by RNA Pol III. The U6 or H1 Pol III promoters are commonly used when generating lentiviruses for shRNA studies.

Multiplicity of infection, number of genomic or infectious particles per cell. The optimal lentivirus MOI varies by cell line. For each cell line, test a range of MOIs from 5 to 100 using positive lentiviral control particles containing a fluorescent protein marker, such as uBriGene's GFP control lentivirus.

Lentivirus can accommodate large packaging capacity and stably integrate into the host genome for long-term gene expression. Most recombinant lentiviruses are Pseudotyped with VSV-G envelope protein, which enables broad cell tropism.

Our lentiviral packaging system is 3rd generation; we can accommodate 2nd generation lentivirus packaging services if client brings packaging plasmids.

uBriGene provides 3rd generation lentiviral packaging plasmids complimentary with our lentivirus packaging services. We also have off-the-shelf GMP-grade lentiviral packaging plasmids with activated DMF.

Client needs to provide transfer plasmid or GOI sequence, uBriGene can provide molecular cloning into a lentiviral transfer plasmid backbone. For larger scale lentivirus packaging services, more plasmid DNA can be provided via our plasmid production service platform.

Lentiviruses have a complex genome with long terminal repeat sequences (LTRs) at both ends. Lentiviral genome contains three structural genes—gag, pol, and env—and six regulatory genes: tat, rev, nef, vif, vpr, and vpu. The gag gene encodes core viral proteins such as the nucleocapsid, matrix, and capsid proteins. The pol gene encodes enzymes essential for viral replication, while the env gene encodes viral envelope glycoproteins. The six regulatory genes are crucial for viral packaging and regulation.