Lentivirus Biology: Insights, Innovations, and Applications

Molecular biology serves as the basis for various biotherapeutics, including recombinant

enzymes (e.g., factor IX monoclonal antibodies (e.g., trastuzumab), and growth factors (e.g.,

erythropoietin). Gene therapy, involving the administration of DNA encoding a desired gene

into cells to treat diseases, extends the reach of molecular biology to potentially alter the

genetic deformities (e.g., β-thalassemia due to β-globin gene defects). Notable examples

include adoptive cellular therapy using genetically engineered T cells, which, via synthetic

genes like chimeric antigen receptors (CARs) or cloned T-cell receptors (TCRs), gain the

ability to target antigens not recognized by their native TCRs. This approach has shown

promising clinical responses, even in patients with high B-cell malignancies.

Gene therapy via lentiviruses, gammaretroviruses, adenoviruses, and adeno-associated

viruses has gained attraction due to viruses' innate ability to transferr genetic material into

cells. Gammaretroviruses and lentiviruses, both retroviral subtypes with an RNA genome

converted to DNA by the viral enzyme reverse transcriptase in transduced cells, are

particularly noteworthy. While gammaretroviral vectors are more commonly used, especially

in research, the number of clinical trials useing lentiviral vectors is increasing.

Lentivirus Genome

Gag, pol, and env are the essential genes for the survival and function of retroviruses and

lentiviruses. Gag encodes structural proteins; pol encodes enzymes necessary for reverse

transcription and integration into the host cell genome; and env encodes the viral envelope

glycoprotein. Retroviruses share a similar life cycle, beginning when the mature virus enters

the cell either through direct membrane fusion or receptor-mediated endocytosis, facilitated

by the binding of envelope glycoproteins to receptors on the cell's surface. Following this

fusion, the virus undergoes uncoating, during which several viral proteins, including some

Gag subunits, dissociate from the viral core. The viral RNA is then converted into proviral

double-stranded DNA through a complex process of reverse transcription. Subsequently, the

proviral DNA complexes with viral proteins to facilitate nuclear import and integration into

the host genome, a process assisted by crucial viral proteins like integrase and host cell

transcription factors such as LEDGF.

In the case of wild-type lentivirus, the integrated proviral genome relies on host machinery to

initiate and complete the transcription and translation of viral proteins necessary for

assembling infectious particles. These viral progenies exit the cell through a process called

budding, wherein virions are released into the extracellular space from the plasma membrane,

distinct from the budding process of other viruses. Lentiviruses, like many enveloped viruses,

utilize the endosomal sorting complexes required for the transport pathway to execute this

complex budding process and release virions into the extracellular space. During budding,

endogenous membrane proteins present within the host cell, such as MHC molecules, can be

incorporated into the virion envelope, potentially influencing the disposition of liberated viral

particles.

The retroviral life cycle involves two unique steps, reverse transcription and integration,

which are fundamental to the functioning of lentiviral vectors. After uncoating, the remaining

viral nucleic acid and protein complex form, which is known as the reverse transcription

complex (RTC). This RTC undergoes a series of steps to convert the single-stranded RNA

genome of the virus into double-stranded DNA.

Reverse transcription begins with a transfer of RNA binding to the primer-binding site at the

5' end of the viral RNA genome. As the negative strand of viral DNA is synthesized by the

polymerase activity of the reverse transcriptase enzyme, the viral RNA is degraded by the

RNase H activity of the same enzyme. This process develops a short fragment of negative-

sense, single-strand DNA, termed strong-stop copy DNA (sscDNA), which then serves as a

primer for the synthesis of the negative-strand viral DNA.

Multiple priming steps lead to the synthesis of a complete DNA copy of the viral RNA, with

an identical U3RU5 sequence within the long terminal repeat (LTR) at both ends. As reverse

transcription progresses, the complex transitions into a preintegration complex (PIC). During

this process, flaps of overlapping positive-strand DNA may form, potentially repaired by host

DNA repair enzymes post-integration. While the central polypurine tracts (or the flaps of

DNA they generate) are not essential for reverse transcription, their presence appears to

enhance the potency of viral vectors, possibly by accelerating second-strand synthesis and

protecting the RTC from innate restriction factors like APOBEC. Additionally, it's suggested

that the three-stranded DNA flap may enhance nuclear import. However, whether reverse

transcription is completed in the cytoplasm or nucleus remains unclear.

The specific composition of the reverse transcription complex (RTC) may enhance the

efficiency of reverse transcription. The capsid seems to shield the viral nucleic acid from

innate sensors while also facilitating more accurate reverse transcription with fewer errors. In

cell lines, reverse transcription is more precise compared to cell-free systems with a slower

rate (70 nt/min vs. 1000 nt/min). This rate further decreases in primary cells like

macrophages and resting T cells due to lower nucleotide pools.

Integration involves several steps, including tethering, 3' processing/cleavage, strand transfer,

and DNA repair. Notably, differences between gamma retroviruses (e.g., murine leukaemia

virus [MLV]) and lentiviruses (e.g., human immunodeficiency virus [HIV]) have certain

implications for designing retroviral gene therapy vectors. Gamma retroviruses like MLV

display a preference for insertion near transcriptional start sites, while lentiviruses like HIV

favor insertion within transcriptional units. Lentiviruses, with their ability to cross intact

nuclear pores, have additional integration criteria related to this unique capability, unlike

gamma retroviruses, which rely on nuclear envelope disassembly during mitosis.

Furthermore, host proteins within the nuclear pore, such as Nup153, Nup98, Nup358, CPSF6,

and TPR, are implicated in facilitating HIV's import. Studies suggest that the capsid protein

of HIV, but not MLV, enables HIV's translocation across the nuclear pore. In non-dividing

cells, integration site selection is influenced by proximity to the nuclear pore, with

heterochromatin typically associated with the nuclear envelope and actively transcribed genes

closer to the pore.

Host proteins LEDGF, BAF, and HMG are all associated with the pre-integration complex

(PIC) of HIV-1. LEDGF interacts with the PIC and epigenetic marks (H3K36me3),

facilitating integration within transcription units. These distinctive characteristics of

lentiviruses could be advantageous for gene therapy platforms targeting quiescent cell types

like long-term hematopoietic stem cells (HSCs).

Traditionally, it was believed that HIV couldn't infect quiescent CD4+ T cells in the G0 stage

but could infect activated CD4+ T cells. However, studies showed HIV DNA in quiescent

CD4+ T cells, suggesting it might originate from previously infected activated T cells

returning to a resting state. Although quiescent CD4+ T cells express HIV fusion receptors,

they exhibit resistance to infection due to factors like limited nucleotides and high expression

of restriction factors, slowing down reverse transcription. Despite this, integrated proviruses

can accumulate in resting CD4+ T cells over a longer period of time compared to activated T

cells.

Another challenge for lentivector infection of resting cells is the inefficient fusion of

quiescent CD4+ T cells with the commonly used viral envelope protein, vesicular stomatitis

virus glycoprotein (VSV-G). VSV-G receptor-mediated endocytosis isn't efficient in resting

CD4+ T cells, but treatment with certain cytokines like interleukin 7 can overcome this

limitation and enhance cell survival.

Lentiviral vectors

The first-generation lentiviral vectors comprised a substantial portion of the HIV genome,

encompassing the gag and pol genes, along with various additional viral proteins. To

facilitate cell entry, these vectors incorporated the envelope protein from another virus,

typically VSV-G, recognizing a widely expressed receptor identified as the low-density

lipoprotein (LDL) receptor. The VSV-G gene was housed separately from the lentiviral

genes. First-generation vectors included accessory and regulatory genes like vif, vpr, vpu,

nef, tat, and rev, which conferred survival benefits for viral replication in vivo but were

dispensable for in vitro growth. Safer, second-generation vectors were subsequently

developed, lacking these accessory virulence factors.

Further safety improvements were made with third-generation lentiviral vectors, which

fragmented the viral genome into separate plasmids to reduce the likelihood of recombinant

virus generation. In this system, the gag and pol genes were on a separate plasmid from the

rev or env genes, resulting in a vector assembled from three separate plasmids containing the

necessary viral sequences. The tat gene was replaced in third-generation vectors by a

constitutively active promoter in the upstream LTRs of the transgene construct. Self-

inactivating (SIN) lentiviral vectors, created by introducing deletions into the 3'LTR of the

viral genome, further enhance safety by disrupting LTR promoter/enhancer activity.

The choice of internal promoters in third-generation SIN lentiviral vectors is important, with

variations observed in their activity across cell types. Although most gene therapy approaches

activate T cells to divide before transduction, lentiviral vectors possess hypothetical

advantages in transducing non-dividing T cells, potentially retaining functional potential.

This targeting strategy may also reduce oncogenic potential compared to vectors targeting

actively dividing cells.

Clinical data indicate that new generation lentiviral vectors significantly reduce the risks of

insertional mutagenesis, with no reported cases of leukemogenesis in gene therapy trials

involving genetic modification of either HSCs or non-dividing T cells. While expansions of

cells with a common integration site have been observed, they do not appear to be related to

oncogenic selection. Lentiviral vectors have shown a lower risk of insertional oncogenesis

compared to gammaretroviral vectors in model systems, with SIN lentiviral vectors

demonstrating diminished genotoxic potential in clinically relevant mouse models.

Clinical Applications and Challenges of Lentivirus Vectors

Lentiviral and retroviral vectors are important technologies undergoing development for

various clinical applications requiring genetic material transfer. Lentiviral vectors have

gained prominence due to their efficiency in transducing non-proliferating or slowly

proliferating cells, such as CD34+ stem cells. The initial clinical use of lentiviral vectors

involved a conditionally replication-competent vector encoding an antisense RNA targeting

the HIV envelope gene, utilized for transducing mature peripheral blood T cells to treat

natural HIV infection. Integration site analysis demonstrated the expected preferential

integration within transcribed genes, with no significant alteration in integration site

distribution between pre-infusion cellular products and engrafted T cells. Lentiviral vector-

based gene transfer into CD34+ HSCs has since been applied in treating various genetic

diseases without adverse events reported in these trials. Notably, in a study transducing HSCs

with β-globin in β-thalassemia patients, one patient achieved independence from transfusion,

associated with an increase in a dominant myeloid clone bearing a lentiviral vector insertion

within the HMGA2 gene locus. The significance of this insertion within the dominant clone

remains uncertain.

Advances in cancer immunotherapy using genetically modified T cells, particularly CAR T-

cell therapies, have shown promising clinical responses in patients with B-cell malignancies.

Clinical trials utilizing CD19-targeted CAR T-cell therapy have demonstrated high efficacy

rates, with some safety concerns noted, and are primarily mechanism-based rather than

vector-related.

One notable on-target side effect of CD19 CAR T-cell therapy is B-cell aplasia, associated

with long-term CAR T-cell persistence. Lentiviral vector-modified T cells have shown

capability in persisting and inducing continued B-cell aplasia for extended periods following

treatment. Additionally, individuals receiving lentiviral vector-based gene therapies may yield

positive results on certain HIV testing platforms, necessitating careful differentiation from

natural HIV-1 infection.

Apart from ex vivo cell modification, lentiviral vectors are also being explored for direct in

vivo therapeutic applications. Clinical studies utilizing non-primate lentiviral vectors for local

gene delivery into the central nervous system and the eye have shown safety and potential

clinical benefit. However, challenges such as efficiency, tissue-restricted promoters, and

immunogenicity need to be addressed for the successful in vivo application of lentiviral

vectors. Surface engineering and genome editing of packaging cells hold promise for

enhancing the stability and efficacy of lentiviral vectors for in vivo use.

Ongoing research endeavors aim to enhance gene therapy efficacy through innovative viral

vector designs. Non-integrating lentiviral vectors (NILVs) have emerged as a promising

strategy to circumvent insertional mutagenesis risks. NILVs lack the viral integrase protein,

enabling transduction of both dividing and non-dividing cells while maintaining the viral

genome as an episome rather than integrating into the host genomic DNA. While the non-

integrating nature of NILVs may result in short-lived gene expression in dividing cells, this

transient expression could be advantageous in certain contexts, such as CAR T cell therapy,

where prolonged expression may not be required.

NILVs have demonstrated effectiveness as a vaccination approach in preclinical models,

eliciting cellular and humoral immunity along with anti-tumor responses. Additionally,

NILVs can be co-transduced with zinc finger nucleases to facilitate DNA recombination with

specific sites in the host DNA. Preclinical studies have shown the potential of zinc finger

nucleases to replace the endogenous T cell receptor (TCR) with a tumor-specific TCR

delivered by NILVs, thereby enhancing safety by mitigating off-target activity.

To enable long-term expression in dividing cells, a dual NILV vector system incorporating

the integrase of phage phiC31 has been developed. Lentiviral vectors have also been explored

in cancer vaccine development, including dendritic cell and cancer cell vaccines. These

vectors have been utilized to express tumor antigens in dendritic cells or modify

costimulatory signals to enhance efficacy. Additionally, lentiviral vectors have been

employed to activate the MAP kinase pathway in dendritic cells, resulting in enhanced anti-

tumor responses in murine models.

Another vaccine strategy involves using cancer cells expressing tumor antigens as the

vaccine. Lentiviral transduction of B-cell lymphoma cells with co-stimulatory proteins and

interleukin-12 demonstrated enhanced immunogenicity in murine models. Lentiviral vectors

have also been used to convert erythroleukemic cell lines into artificial antigen-presenting

cells for T cell expansion and potential in vivo vaccination.

Furthermore, lentiviral vectors have been utilized to deliver components of gene editing

systems, such as guide RNAs for the CRISPR-Cas9 system. These vectors enable precise

gene editing in target cells, offering potential alternatives to genetic screening methods.

Concerns about off-target effects have prompted the exploration of approaches to limit the

duration of gene editing, including the co-expression of guide RNAs targeting the Cas9

nuclease itself for degradation. Despite the early stage of development, lentiviral vectors

present a versatile platform for delivering gene editing tools and altering gene expression,

offering potential avenues for novel therapeutic interventions.

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