Lentiviral vectors for CRISPR-mediated gene editing (Part 40- CRISPR in gene editing and beyond)
Welcome to the 40th part of the multi-part series on applications of CRISPR in gene editing and beyond.
The third type of viral vectors used for CRISPR-mediated gene editing purposes are lentiviral vectors, derived from human immunodeficiency type-1 (HIV-1) lentivirus.
The third type of viral vectors used for CRISPR-mediated gene editing purposes are lentiviral vectors, derived from human immunodeficiency type-1 (HIV-1) lentivirus. Lentivirus is a type of retrovirus that can infect both dividing and non-dividing cells. It is an RNA virus because its genome is made up of ssRNA, while its host’s genetic material is in the form of DNA.
When a lentivirus infects a host cell, it transfers its RNA genetic material along with specific enzymes, namely reverse transcriptase and integrase, into the host cell. The virus then undergoes a process called reverse transcription, where it creates a DNA copy from its RNA molecule using the reverse transcriptase enzyme that it carries with it. After this, the produced DNA is incorporated into the host cell genome with the help of another enzyme carried in the virus called integrase.
Structurally, the lentivirus genome contains 3 major genes: gag, pol, and env genes, flanked by long terminal repeats (LTRs) on both the 5’ and 3’ ends (Fig 1a). The gag gene encodes for the viral core proteins like matrix, capsid, and nucleocapsid. The pol gene encodes for the reverse transcriptase, integrase, and protease enzymes, which are crucial for reverse transcription and genomic integration. And the env gene encodes for the surface glycoproteins gp120 and gp141 that are responsible for binding to receptors on the surface of target cells and facilitating virus entry into the cell (Fig 1b).
Within the lentivirus genome are two regulatory genes, tat and rev, which facilitate viral transcription. Additionally, the genome carries four accessory genes — vif, vpr, vpu, and nef that contribute to HIV virulence, although they are not as critical to viral replication in host cells. The genome also contains Ψ sequence, which is required for the packaging of viral particles. And the long terminal repeat sequences present on both ends of the genome help the reverse transcribed DNA to integrate into the host genome.
In the past two decades, these properties of the genetic material of lentiviruses have turned them into an attractive system for use as vectors for transferring genes into mammalian cells. But before using the HIV viruses as vectors, its genome must be modified extensively to be safe for use in gene editing or gene therapy purposes.
To construct a safe and effective lentiviral vector, the accessory HIV virulence genes vif, vpr, vpu, and nef are removed entirely. And the other genes necessary for viral infection gag, pol, env, and rev are provided in trans, meaning these genes are expressed in different plasmids. For instance, 3 types of helper plasmids are used for the expression of different genes.
(i) Helper plasmid 1 contains gag and pol genes (Fig 2a).
(ii) The second helper plasmid contains the 5’ and 3’ long terminal repeats and an env gene (Fig 2b).
(iii) And the third helper plasmid contains the rev gene that is important for exporting viral mRNA from the nucleus to the cytoplasm during infection (Fig 2c).
Each of these plasmids drives its gene expression from the well-studied promoters from other viruses such as cytomegalovirus, abbreviated as CMV, or respiratory syncytial virus, abbreviated as RSV.
These plasmids also contain additional genetic elements such as polyadenylation sites polyA that are important for the stability of mRNA molecules transcribed from these plasmid constructs.
The envelope glycoprotein of HIV, gp120, specifically binds to the CD4 receptor on the surface of CD4 T cells, which are a type of immune cell. This specificity of envelope glycoproteins narrows the host cell range of HIV vectors and limits the usefulness of HIV lentiviral vectors for gene editing and gene therapy purposes. To overcome this limitation, the env gene in the helper plasmid 3 is replaced with an envelope glycoprotein from another virus called the vesicular stomatitis virus, which is known as the VSV-G protein. The VSV-G protein interacts with phospholipids rather than specific receptor molecules on the target cell, allowing the lentiviral vector to infect a broader range of cell types.
Apart from 3 helper plasmids, there is a fourth plasmid that contains a transgene with the promoter, 5’ and 3’ long terminal repeat sequences for the genome integration into the host DNA and the packaging signal Ψ sequence (Fig 2d). The insert capacity of the lentiviral vector is 8 kb of sequence.
The combination of 4 vectors is known as the lentiviral gene transfer system. When these plasmids are introduced into the packaging cell lines by a process called transfection, the transgene vector integrates into the cell line DNA and produces mRNA (Fig 3).
The other three plasmids also produce mRNAs which are translated to form viral proteins, including the capsid, reverse transcriptase, and envelope proteins. These proteins assemble to form the viral particles, which package the mRNA containing the transgene. Once the lentiviral particles are released from the packaging cell lines and infect the target cells, the mRNA containing the transgene is converted into DNA by reverse transcriptase; the new DNA enters the host cell’s nucleus and integrates into host chromosomal DNA (Fig 4). The integrated DNA contains LTR and Ψ sequence, but none of the other components from the HIV genome. This means that the integrated DNA cannot form viral particles, as it lacks the other necessary viral components. However, the transgene can be expressed and produce functional proteins in the target cells.
Lentiviral vectors for CRISPR-mediated gene editing
For lentivirus-mediated CRISPR-Cas9 gene editing, two transgenes are required that code for gRNA and Cas9 enzyme. The two transgenes along with their promoters U6 are inserted into the transgene-containing vector. When the viral particles containing transgenes gRNA and Cas9 as their genome are allowed to infect the target cells, gRNA, and Cas9 genes are expressed and perform gene editing at the desired position in the genome.
One of the challenges of using lentiviral vectors for gene editing is the potential for random integration of the transgene into the host genome, which can lead to unwanted effects such as disruption of endogenous gene function or activation of oncogenes.
To address this issue, non-integrating lentiviral vectors have been developed. These vectors are engineered to eliminate the activity of the integrase enzyme, which is responsible for integrating the transgene into the host genome. This is typically achieved through specific mutations in the integrase coding region, which disable its activity without affecting other important functions such as reverse transcription and mRNA transport.
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