In the pursuit of a cure for AIDS, scientists have devoted great efforts into studying the
HIV lentivirus. Although a cure for AIDS remains to be seen,
research efforts into the HIV lentivirus have led to the development of powerful HIV-based
tools for transgene delivery. Today, lentiviral vectors are major tools
for basic and translational research, enabling scientists to stably overexpress or silence
genes, immunize against a range of diseases, generate transgenic organisms, and much more. The Lentivirus is part of the Retroviridae
family of viruses, with HIV-1 being the most widely studied. Members of this family contain positive single
stranded RNA that is reverse-transcribed into DNA and integrated into a host cell's genome. The HIV Lentiviral RNA genome contains three
major genes: gag, pol, and env. The gag gene encodes protective core and matrix
proteins for viral assembly and infection. The pol gene encodes for reverse transcriptase,
RNase H, and integrase, enzymes crucial for reverse transcription and genomic integration. The env gene encodes for surface glycoproteins
that determine tropism and enable entry into the cell. The Lentivirus genome also contains two important
regulatory genes, tat and rev, that activate viral transcription, as well as 4 accessory
genes, vif, vpr, vpu, and nef, that are less essential for virus replication in host cells. Lentiviruses have a lot of potential as tools
for gene delivery and gene therapy. This is because
their stable integration into the host cell genome ensures long term expression for stable cell
line generation. They can infect both dividing and non-dividing
cells such as hepatocytes and neurons, cells that are notoriously difficult to infect. They have broad tropism, making them effective vehicles for delivering CRISPR sgRNA
constructs to edit the genes within any tissue type. They don't generate immunogenic proteins, and
they can deliver transgene fragments as large as 9 kb. Because of all these advantages, great efforts have been put into developing recombinant
lentiviruses for clinical and research use, due to its robust infection ability. However,
precautions are needed to ensure replication-competent lentiviruses are not accidentally generated
via recombinations between the delivered as well as endogenous viral elements in producer
cells. In an effort to reduce biosafety risk, three
generations of recombinant lentiviral packaging systems have been developed, with the first
generation system no longer in use due to biosafety risks. The second generation system splits essential
components of the lentiviral system across three plasmids that are delivered separately
for safety. The transfer plasmid encodes for the transgene. It also contains cis-acting elements such
as the 5' and 3' LTRs essential for promoting RNA polymerase II to begin transcription of
viral mRNA, and the psi sequence which signals genome packaging. The packaging plasmid is provided in trans
and encodes only the essential trans-acting genes gag, pol, rev, and tat that are required
for entry and integration of the viral genome. The envelope plasmid contains genes encoding
for envelope proteins. For safety reasons, all other genes involved
in viral propagation, for example, vpr, vif, vpu, and nef, are excluded. To almost completely eliminate dangerous lentiviral
recombination events, an even safer, third generation system was created. In this system the viral genome is split into
four plasmids: a plasmid containing only the packaging genes
gag and pol, a plasmid with only the regulatory gene rev,
a plasmid carrying only the envelope gene env, and
a transgene plasmid. In addition, the LTRs flanking the transgene
are further modified as they contain enhancer and promoter regions that can activate adjacent
cellular proto-oncogenes. By removing this enhancer and promoter region, U3, from the 3' LTR, a replication incompetent "self-inactivating" (SIN) system is created as this deletion is transferred to the 5' LTR after reverse transcription and integration. The U3 of the 5' LTR is also replaced with
a CMV promoter to eliminate the need for the transcriptional transactivator, Tat. Although the third generation system is significantly
safer, its viral yield is typically lower than the second generation system. At abm, we offer both the second and third
generation lentiviral packaging systems to suit any application. Finally, as tropism is determined by the glycoproteins on the viral surface, a lentivirus' tropism
can be further broadened by replacing the HIV-1 env glycoprotein with the envelope glycoprotein
G from the vesicular stomatitis virus (VSV-G). VSV-G is incorporated in all abm lentivirus
packaging systems to widen the range of cell types the virus can bind to. Recombinant Lentiviral Systems show great promise as potent tools for gene therapy to
treat HIV infection, cancer, Parkinson's Disease, and many more. A breakthrough is
surely not far on the horizon. In the meantime, recombinant lentiviral systems
have secured their place as an invaluable tool for advancing in vitro and in vivo research
across a variety of areas in biology and life sciences. From siRNA lentivectors to CRISPR Cas9 lentivectors
to Cumate-inducible lentivectors, our lentiviral vectors and ready-to-use lentiviruses can
be used to manipulate gene expression of any human, mouse, or rat gene. Simply select your gene of interest, as well
as a promoter, tag, and/or reporter and we will package it for you at the titer you desire. Check in with our store at www.abmgood.com
and explore our catalogue of lentivirus products and services today! If you enjoyed our video, please comment and
subscribe to our channel. To learn more about the Lentivirus System, visit our Lentivirus Knowledge Base. As always, if you have any questions, leave
them below and we'll get back to you shortly. Thank you for watching!
