- Lentivirus came from HIV
- Which generation is this?
- Why bother titering?
- What are some different ways to titer?
- What makes lentivirus so special?
- What is pseudotyping?
- Practical applications and limitations
Lentivirus came from HIV
HIV is part of a subfamily of retroviruses called Lentivirus. HIV derives its virulence from 9 genes: Env, Vif, Vpr, Vpu, Nef, and Tat. Of the nine genes, six can be removed without altering HIV’s gene transfer ability. Lentiviral vectors are created by stripping down the HIV genome to the “bare bones,” breaking the provirus into pieces, and leaving the vectors replication defective. What remains are 3-4 plasmids and a safer method for transfection:
- Lentiviral Backbone or transfer vector plasmid: contains gene of interest/shRNA
- Packaging Plasmid
- Envelop Gene Plasmid
Which generation is this?
Lentivirus vectors have evolved over time to achieve higher efficiency and biosafety. Lentiviral packaging particles are primarily made from 3 components: genomic RNA, internal structural and enzymatic properties, and the envelope glycoprotein.
- 1st Generation is composed of all HIV genes minus the envelope protein.
- 2nd Generation has 5 of the 9 genes deleted leaving only gag/pol and the tat/rev regions. These regions are responsible for structural and enzymatic components and transcriptional and post-transcriptional genes respectively.
- 3rd Generation is geared towards clinical applications and is considered the safest method. Third generation packaging systems contain only gag, pol, and rev genes. It utilizes a chimeric 5’ LTR to ensure transcription in the absence of Tat.
In higher generations, the transfer vector (Viral Backbone) contains the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) to increase levels of transcripts. The transfer vectors are also designed to be self-inactivating. This is achieved by removing the U3 region of the 3’ LTR which results in the virus being unable to reconstitute its promoter.
It is important to understand which generation your transfer vector is because the vector must be packaged with the corresponding generation of packaging vectors. For instance, a 2nd generation Lentivirus backbone can not be packaged with3rd generation packaging vectors.
Why bother titering?
Infection-based titer values are the most reliable means to calculate actual infectious particles, although it can under-estimate actual values. Titers calculated from lower dilutions represent a more accurate value for virus particles present, although this number may not be a practical working value for experimentation. Use the titer values to provide consistency among your experiments.
What are some different ways to titer?
Functional titer is defined as the number of functional vector particles required to infect a cell, present in a volume. There are several methods for measuring titer. Some are more reliable than others. Other methods include: measuring the p24 concentration/ml, measuring RNA equivalents, Transducing units/ml, or measuring mRNA equivalents. The first two are unreliable because they tend to overestimate the vector titer. The amount of p24 measured comes from functional particles, free p24, and nonfunctional vector particles. The RNA assays measure defective particles as well.
Reliable methods are determined by transduction of cells following limiting dilution of vector and subsequent evaluation of reporter protein activity (eGFP) or by the number of colonies following antibiotic selection.
The most straightforward method is to quantify functional vector titer by employing fluorescence and FACs. This is the method employed by the ViraCore. The method does have some limitations including being restricted to fluorescent reporter proteins and it cannot distinguish between single or multiple integrations. This method is best because it only accounts for functional particles. Titers are calculated from infection of live cells in a 96 well plate. The formula used is: (#cells X %infected cells) X 1,000 μl/virus (ml). It is reported in TU/ml which translates to Transducable Units per milliliter.
What makes lentivirus so special?
Though there are several virus delivery systems available today for gene therapy, lentivirus is considered one of the best methods. Several features make lentivirus preferable especially the ability to infect both dividing and non-dividing cells. Lentiviral vectors can integrate their genetic cargo directly into the chromosome of the target cell but do not transfer sequences that encode for proteins derived from the packaging virus. This is key to preventing an immune response to the cells containing the transfer gene. Finally, lentivirus vectors can be pseudotyped to infect specific or a broad range of targets.
What is pseudotyping?
In HIV, pseudotyping is used to change the tropism of the virion. In other words, pseudotyping changes the way the Lentivector targets a specific cell for infection. In wild type HIV-1, the envelope protein recognizes and binds CD4, a molecule present of the surface of helper T cells and other immune cells. Most often, the HIV envelope protein is replaced with vesicular stomatitis virus glycoprotein-g (VSV-G). It is commonly used because it brings stability to the lentivector allowing it to be ultracentrifuged, but also because its phospholipid receptor is universally expressed in mammalian cells. Due to its broad tropism, VSV-G is a good choice for pseudotyping HIV lentivectors. However, lentivectors can be pseudotyped to infect specific cells or cell types. Using glycoproteins that have select tropism could be used to infect specific targets. For instance, glycoproteins derived from viruses that cause lung infection could be useful for gene therapy of the human airway.
Practical applications and limitations
Applications of lentivirus vectors include stable gene transfer for gene delivery in gene and cell therapy. This includes using RNA interference technology to block the expression of specific genes. Along with RNAi, lentivirus can be used to introduce a new gene into human or animal cells. These applications are possible because of lentivirus vector’s ability to stably transfer genes, long term expression, and the ability to specifically infect cells by pseudotyping.
Lentivirus vectors do have some limitations such as a (somewhat) limited size constraint. Lentivirus vectors can package large gene inserts with a sharp titer decline after 10kb total proviral size. Typically a gene cloned into a transfer vector produces the highest titer virus when the final size is equal to or smaller than the WT HIV-1 genome (9.7kb).