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For the past 25 years, tremendous efforts have been put into understanding the structure and biology of the HIV virus in the hopes of curing AIDS. In the pursuit of a cure, potent HIV-based transgene delivery vectors were developed (1). Although lentiviral vectors can also be derived from other primate lentiviruses (HIV-2 and SIV) and non-primate lentiviruses (MVV, FIV, EIAV, CAEV and BIV), HIV-based vectors make up the majority of lentiviral vectors used in research today. Due to its unique advantages and the remarkable improvements made upon the recombinant lentivirus system, lentiviral vectors are now widely used in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, transgenic animals, stem cell modifications, and much more (2).

The lentivirus belongs to the retroviridae family of viruses. The retroviridae genome consists of a single stranded positive sense RNA that is converted into double stranded DNA during the replication process. In most other types of viruses, viral DNA is transcribed into RNA which is then translated into protein. In contrast, retrovirus RNA is first reverse-transcribed into DNA and then integrated into the host cell genome. After integration, the host cell will transcribe the viral genes along with its own genes, thereby producing stable transgene expression. In addition, lentiviruses are unique members of the retroviridae family as most retroviruses cannot productively infect non-dividing cells (3) whereas lentiviruses can infect cells regardless of their proliferation status (4), making them particularly attractive for human gene therapy. Hepatocytes, neurons, hematopoietic stem cells, monocytes, and macrophages are some examples of potential targets for lentiviral vector based gene therapy (5).

HIV-1 is the most well-studied and used lentivirus in research (5). HIV has a single stranded RNA genome of around 9 kb in length that includes three major structural genes: gag, pol, and env. (Figure 1)

Gag is first transcribed into unspliced mRNA and then cleaved into transcripts encoding the three viral core proteins:.

• matrix (MA) proteins, which are necessary for virion assembly and infection of non-dividing cells;
• capsid (CA) proteins, which form the hydrophobic core of virion
• nucleocapsid (NC) proteins, which protect the viral genome by coating and associating tightly with viral RNA in virions

The pol gene encodes for the viral protease (PRO), reverse transcriptase (RT) and integrase (IN), enzymes essential for viral replication.

The env gene encodes the viral surface glycoprotein gp160, which is cleaved into the surface protein gp120 (SU) and transmembrane protein gp41 (TM) during the process of viral maturation. These surface proteins are essential for virus entry into the host cell as they enable binding to cellular receptors and fusion with cellular membranes (5).

In addition to these major genes, the viral genome also contains regulatory genes (tat and rev) as well as 4 accessory genes (vif, vpr, vpu, and nef). Tat encodes the transactivators critical for activating viral transcription while rev encodes a protein that regulates the splicing and export of viral transcripts. Tat and rev are the first proteins to be synthesized following integration and are required to accelerate the production of viral mRNAs. On the other hand, the proteins encoded by accessory genes are not essential for virus replication in host cells (5). Among them, vpr is a virion-associated protein present only in primate lentiviruses. Vpr can act as a weak transcriptional transactivator of the long terminal repeats (LTR) present in the viral genome (6) and participates in enabling infection of non-diving cells (7).

As mentioned, two LTRs flank the viral genome and are required for viral transcription, reverse transcription, and integration of the viral genome. Located between the 5’-LTR and the gag gene is another critical component, ψ, which is a signal for genome dimerization and packaging (Figure 1).

The Lentivirus lifecycle can be summarized by six major steps:

• Binding and Entry
• Uncoating
• Reverse Transcription
• Proviral Integration
• Viral Protein Synthesis and Assembly
• Budding

In the first stage, the lentivirus binds to its target cell via interactions between its viral envelope glycoprotein and a specific cell surface receptor which determines the cellular target for the virus (viral tropism). The natural HIV infection cycle is initiated by the attachment of glycoprotein (SU) to its primary receptor CD4 as well as its co-receptor CXCR4 (expressed on T-lymphocytes) or CCR5 (expressed on monocytes). Upon receptor recognition, viral transmembrane proteins (TM) change conformation to facilitate membrane fusion of HIV with the host cell, leading to viral entry.

After entering the cell, the virion-bound matrix and capsid proteins disassemble, releasing the viral genome as well as viral proteins such as matrix (MA), reverse transcriptase (RT), integrase (IN), and Vpr proteins into the cytoplasm. Using the viral RNA as a template and host nucleotides, the reverse transcriptase synthesizes viral DNA, henceforth referred to as pro-viral DNA. The pro-viral DNA is then imported into the nucleus and integrated into the host genome via the action of viral integrase (8).

Following the integration of the pro-viral DNA, the LTR at the 5’-end of the viral genome acts as a combined enhancer and promoter, enabling the host cell’s RNA polymerase II to begin transcription of the viral genome. The LTR at the 3’-end of the genome stabilizes newly synthesized transcripts by regulating their polyadenylation. Initially, the basal promoter activity of the 5’-LTR is minimal and the initial transcription is inefficient. These first viral mRNAs are multiply spliced into short transcripts encoding the non-structural proteins Tat, Rev, and Nef, proteins that facilitate the production of other viral transcripts necessary for progression through the viral lifecycle. Newly synthesized Tat transactivates and amplifies the transcription of other structural proteins (9) while Rev bind to RRE ( Rev-responsive element) on the viral transcripts to facilitate nuclear export of singly spliced or non-spliced viral mRNAs (10). Singly spliced transcripts encode Env as well as accessory proteins, whereas non-spliced viral RNAs are translated into Gag and Pol or act as genomic RNAs for progeny viruses.

In the final stage, the exported viral genome and proteins are assembled at the plasma membrane and released from the host cell (11) (Figure 2).

Replication, integration, and packaging of lentiviruses are mediated partially by cis-acting RNA or DNA sequences which do not encode proteins. Cis-acting elements such as the LTRs and ψ signals are essential in the design of recombinant lentiviral vectors and are generally included in the transfer plasmid (the part of a lentiviral vector which will integrate into the host cell genome and encode targeted genes).

The trans-acting viral elements encode structural, regulatory, and accessory proteins. Recombinant lentiviral vectors are deprived of their replication abilities for safety reasons. In the recombinant lentiviral vector, only the genomic components necessary for the early steps of the lentivirus life cycle are included (binding and entry, uncoating, reverse transcription, and pro-viral integration). Genomic components that facilitate viral protein synthesis can be excluded from the recombinant lentiviral vector. Only essential trans-acting genes such as gag and pol (necessary for viral reverse transcription and integration) as well as env (necessary for binding to host cells) are preserved.

Generally, the transfer plasmid contains the gene of interest along with the essential cis-acting elements, while essential trans-acting genes are provided separately (in trans) by the packaging plasmids (Table 1).

Table 1 – Important Cis-acting and Trans-acting elements of HIV-1-based lentiviral vectors.

Molecular	Function	Necessity
1. Cis-acting
LTRs	Contain sequences required for viral gene expression, reverse transcription, and integration	Essential
Ψ	Required for packaging of the genomic transfer RNA	Essential
RRE	Rev response element. Required for processing and transport of viral RNAs	Beneficial
2. Trans-acting
gag/pol	Encodes structural proteins and enzymes required for viral reproduction	Essential
env	Encodes envelope glycoprotein	Essential
vif	Assists assembly of the virions and infectivity	Optional
vpu	Assists release of virions	Optional
vpr	Assists infection of non-dividing cells	Optional
tat	Necessary for high level expression of viral LTR	Optional
rev	Necessary for expression of unspliced and singly-spliced mRNAs in vivo	Beneficial
nef	Required for high viral burden	Optional

Lentivirus Pseudotyping

One property common to most retroviruses is the ability to form pseudotypes or retroviral particles that have incorporated a heterologous envelope glycoprotein (12). The envelope glycoprotein G of the vesicular stomatitis virus (VSV-G) is widely used to create pseudotyped retroviral vectors as they offer unique advantages over unmodified vectors. Firstly, VSV-G is much more stable than the natural retroviral or lentiviral envelopes, making it easier to achieve higher viral concentrations by ultracentrifugation (13). More importantly, VSV-G binds the ubiquitous membrane component phosphatidylserine, which enables the VSV-G pseudotyped virus to attach and transduce a much wider range of cells (14). Given the benefits, Akkina et al. (15) replaced the HIV-1 Env glycoprotein with VSV-G and has successfully demonstrated that the VSV-G pseudotyped HIV vector has high efficiency gene transfer into hematopoietic stem cells. Currently, most lentiviral vectors are pseudotyped with VSV-G to enable robust transduction into many cell types.

First generation HIV-1-based recombinant lentiviral vectors (Figure 3A)

Since lentiviral vectors are derived from the HIV-1 pathogen, biosafety issues are an important concern. During the process of using replication-defective viruses, there is a possibility of accidentally generating RCLs (replication-competent lentiviruses) via recombinations between the delivered as well as endogenous viral elements in producer cells.

In an attempt to generate a safe system for laboratory use, the first generation recombinant lentiviral system splits the viral genome into three separate plasmids: (a) a packaging plasmid; (b) an Env plasmid encoding the viral glycoprotein; and (c) a transfer vector genome construct. The packaging plasmid expresses HIV Gag, Pol and regulatory/accessory proteins under the control of a CMV promoter, all of which are required for vector packaging. The Env plasmid expresses a viral glycoprotein such as VSV-G to provide the vector particles with a receptor binding protein. In order to avoid their transmission into vector particles and to reduce the production of RCLs in vector preparations, the packaging signal ψ or the LTRs are not included in the packaging and Env plasmids. Instead, LTRs, ψ, as well as RRE are included in the transfer vector plasmid. Therefore, the transfer plasmid encodes for proteins necessary for packaging, reverse transcription and integration, but not for the expression of HIV proteins. In this way, the genomic components responsible for packaging the viral DNA are separated from the genomic components that activate them. Thus, the packaging sequences will not be incorporated into the viral genome and the virus will not reproduce after it has infected the host cell (16, 17).

Second generation recombinant lentiviral vectors (Figure 3B)

Despite the precautions, the first generation recombinant lentiviral vectors are no longer commonly in use due to biosafety risks. This prompted the development of a safer, second generation recombinant system. In this system, the genomic components encoding viral accessory proteins (Vif, Vpu, Vpr or Nef) are removed. These accessory proteins are important for HIV propagation in primary cells or in vivo (18), but not essential for lentiviral vector functions. The second generation recombinant system therefore includes only four of the nine HIV genes: gag, pol, tat, and rev (19).

Third generation recombinant lentiviral vectors (Figure 3C)

Traditional lentiviral vectors integrate transgene cassettes flanked by the two LTRs into the host genome. However, the existence of intact LTRs in the host genome gives rise to potential safety risks. Firstly, if replication-competent recombinant lentiviruses are produced accidently, they can replicate as the wild-type viruses. In addition, the vector-transduced cells may be infected by a wild type lentivirus which can potentially act as a helper virus to rescue the integrated vector into new viral particles for replication. Finally, because LTRs have an enhancer (a binding site for host transcription factors) as well as promoter region, integration of LTRs into the genome can activate adjacent cellular genes that may be proto-oncogenes. Given these concerns, SIN (self-inactivating) lentivectors were developed.

In the third generation self-inactivating recombinant system, the U3 region of the 3’-LTR is deleted. Originally, each LTR contains three regions: U3, R, and U5, with the U3 the component acting as the enhancer/promoter. If U3 in the 3’-LTR is deleted, the same deletion would be copied and transferred into the 5’-LTR promoter/enhancer region of the integrated genome during reverse transcription. This deletion will result in transcriptional inactivation of potentially packageable viral genome in the transduced cell (20).

Tat and Rev are absolutely essential for HIV-1 replication because they regulate viral transcription and nuclear export of transcripts (21, 22). Thus to significantly increase safety, Tat is deleted from the packaging plasmid of the third generation recombinant system. Tat’s function is instead realized by replacing the U3 promoter region of the 5’-LTR in the transfer plasmid with other strong viral promoters such as CMV or RSV (23). Furthermore, Rev is provided from yet another separate plasmid for added safety.

In a nutshell, the third generation recombinant lentiviral vector system has four plasmids:

• a packaging construct containing only gag and pol genes;
• a plasmid expressing Rev;
• an Env (i.e. VSV-G) plasmid;
• a transgene plasmid driven by a heterologous strong promoter.

Because the vector system is split into four plasmids, at least three recombination events are required to generate a replication-competent HIV-1-like virus. Even if such events occurred, the resulting viruses only have Gag, Pol, Rev and VSV-G proteins, with no active LTRs, Tat or accessory proteins, markedly increasing the safety of the vectors. However, when compared to a three plasmid system (i.e. second generation), the virus yield from a third-generation vector is typically lower.

Lentiviral vectors are regarded as attractive gene-delivery vehicles for several reasons:

• they offer long term gene expression via stable vector integration into host genome;
• they are capable of infecting both dividing and non-dividing cells;
• they are capable of infecting a broad range of cells including important target cell types for gene and cell therapies;
• they lack immunogenic viral proteins after vector transduction;
• they can deliver complex genetic elements such as intron-containing sequences;
• they are a relatively easy system for vector manipulation and production.

Due to these unique advantages, lentiviral vectors are now widely applied in basic and translational science as well as clinical trials. There are many commercially available premade lentiviral vectors, which are engineered to express or silence genome-wide genes (2).

The major disadvantage of lentiviral vectors is the potential for recombination leading to replication competent lentivirus (RCL). Production of RCL in preparations of replication-defective vectors has been recorded in numerous conditions (24, 25, 26). In later generations of vectors in which viral protein coding regions were split in the packaging cells, the frequency of recombination leading to RCL was decreased but not eliminated (27). The application of the SIN property in the vectors dramatically reduces this likelihood (28). Another concern involves the activation of the cellular proto-oncogene by residual promoter activities of integrated LTRs or the transcriptional interference and suppression by LTRs (29). Again, the SIN approach not only minimizes the expression of vector RNA, but also prevents the insertional activation of cellular oncogenes or transcriptional interference by the integrated virus.

As lentiviral infection ability of different cell types varies, different cell types may require different MOIs for successful transduction and knockdown of the target gene. Theoretically, a higher MOI will generate a higher number of transductions per cell, a higher number of transgene integrations, and a higher expression. To determine the optimal amount of lentiviruses needed for efficient transduction of your cell line, it is highly recommended that a range of MOIs (e.g. from 0.1 to 20) be tested. In order to determine the MOI, you will need to know your viral titer, or the concentration of infectious particles (IU). For example, to achieve an MOI of 2, 50 microliters at 108 IU/ml will provide enough particles to transduce 5 wells of cells, with each well containing 5 x 105 cells. Refer to the equation below to help calculate the MOI:

$$MOI = {Viral\; Titer {\left(\frac{IU}{ml}\right)} \times \; Volume \;(ml) \over Total\; number\; of\; cells}$$

You can read more about this topic in our knowledge base article, Multiplicity of Infection (MOI).

The first clinical study using a lentiviral vector was approved in 2001 for anti-HIV therapy (30). In this clinical trial, a VSV-G-pseudotyped HIV-based vector was engineered to conditionally express an antisense RNA against HIV envelope glycoprotein in the presence of regulatory proteins provided by the wild-type HIV virus. The safety and efficacy HIV-1 envelope antisense gene delivery to CD4+ T cells was evaluated. Four out of five subjects with chronic HIV infection showed an increase of CD4+ T cells after receiving a single dose of gene-modified autologous CD4+ T cells. In addition, the viral load decreased in all five patients after 1 year. Further observations over 2 years have not detected any adverse clinical events (31).

In another instance, a Phase I/II clinical trial of β-globin gene therapy for β-thalassaemia began in 2007 (32). In this trial, a self-inactivating lentivector, LentiGlobin, which contains the β-globin gene (including its introns, promoter, and β-locus control region/β-LCR), was used to transduce CD34+ cells ex vivo. These autologous gene-modified cells were then transplanted into patients. After 3 years of treatment, patients demonstrated corrected β-globin gene and stable blood hemoglobin levels (33). No insertional mutagenesis has been reported so far.

Up until July 2015, about 114 clinical gene therapy trials using lentiviral vectors are ongoing or have been approved, including treatment for HIV infection, monogenic diseases (X linked cerebral adrenoleukodystrophy, Sickle cell anemia, Wiskott-Aldrich Syndrome, Metachromatic Leukodystrophy, X-Linked Chronic Granulomatous Disease, Inherited Skin Disease Netherton Syndrome, mucopolysaccharidosis type VII, β-thalassemia, Fanconi Anemia Complementation Group A, X-Linked Severe Combined Deficiency, Adenosine Deaminase Deficient Severe Combined Immunodeficiency, Hemophilia A), various cancers, Parkinson’s disease and many more (34).