February 24, 2024

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How do Viral Vectors-Lentivirus play the role in Gene Therapy?

How do Viral Vectors-Lentivirus play the role in Gene Therapy?


How do Viral Vectors-Lentivirus play the role in Gene Therapy?

Throughout the 40 years of gene therapy, it has made great strides in the treatment of human diseases, bringing hope to patients and families with limited treatment options, but has also suffered many setbacks, serious adverse reactions, and even deaths.


JoteT. Bulcha et al. published a review of “Viral vector platforms within the gene therapy landscape” in Nature, expounding the mechanism of viral vectors in gene therapy and their promotion in the treatment of human diseases.


How do Viral Vectors-Lentivirus play the role in Gene Therapy?

The introduction will be made in three parts: adenovirus, adeno-associated virus and lentivirus. The article shared in this issue is: Lentiviral vector – a vector for transgenic cell therapy.



Lentiviral vector: A powerful vector for transgenic cell therapy

Lentiviruses belong to the retroviridae family and include 8 viruses capable of infecting humans and vertebrates.

The primary infected cells are mainly lymphocytes and macrophages, resulting in the disease of infected individuals.

The main clinical feature of lentiviral infection is that it goes through a long incubation period before the typical clinical symptoms appear, and then slowly develops, so it is called lentivirus. It is often associated with chronic diseases of the immune system and central nervous system, such as human immunodeficiency virus (HIV), which causes AIDS.


1. Lentiviral structure and infection mechanism

Human immunodeficiency virus (HIV) is a complex retrovirus, spherical, enveloped, and single-stranded RNA virus.

The genome size is 9.7kb, and the long terminal repeats (LTR) at both ends consist of U5, U3 and R sequences, which affect viral genome replication.

The cis-acting element ψ (psi), located at the C-terminus of the 5′ LTR, is critical for transmitting the packaging signal of the viral genome. The sequence encoding the protein consists of 9 genes grouped into the following three categories:

(1) The main core protein genes of the virus: gag, pol and env.

① gag gene encodes viral structural proteins such as nucleocapsid protein, inner membrane protein and capsid protein;

② The pol gene encodes enzymes related to viral replication, such as reverse transcriptase and integrase;

③ The env gene encodes the viral envelope glycoprotein, which determines the tropism of the virus.


(2) Genes encoding regulatory proteins: tat and Rev.

① tat supports transcriptional activation and elongation of RNA polymerase II by binding adjacent to LTR;

② Rev coordinates the nuclear export of spliced ​​and unspliced ​​viral RNA by binding to a motif in the env gene region, that is, participates in protein-regulated expression.


(3) Genes encoding accessory proteins: vif, vpr, vpu, and nef, which can increase viral titers.


How do Viral Vectors-Lentivirus play the role in Gene Therapy?
(The picture shows the design of the third-generation lentiviral vector based on HIV-1)


Infectious lentiviral particles enter host cells mediated by interactions between glycoproteins on the outer envelope and specific cellular receptors.

Successful binding to cell surface receptors results in fusion of the virion lipid bilayer with the cell, followed by entry of the viral RNA genome into the cytoplasm.

After reverse transcribing into cDNA, it is randomly integrated into the host genome, and it has a preferential integration to the transcriptional active site.


2. Lentiviral gene therapy vector design and modification

Lentivirus as a vector for gene therapy has the following characteristics:

① Transgenic integration and long-term, high-level expression, packaging capacity up to 9kb;

② Lentiviral vectors can express multiple genes at the same time;

③ Lentiviral vectors can transduce dividing and non-dividing cells;

④ The innate immune response caused by lentiviral vectors is weak.


In order to avoid the production of replication competent virus (RCV), when constructing HIV-1 lentiviral vector, the HIV-1 genome is generally assembled into several plasmid vectors, and then co-transfected into cells, and then only one infection ability is obtained.

Replication incompetent HIV-1 virus particles. At present, the design of lentiviral vectors is divided into the following stages:

(1) The first-generation lentiviral packaging system: with HIV-1 as the backbone, the cis-acting sequence structure required for reverse transcription and integration and the sequence encoding the trans-acting protein are separated to construct packaging plasmids, envelope plasmids, and transfer plasmids respectively. Plasmid, vesicular stomatitis virus (VSV-G) glycoprotein replaces the env gene.


(2) Second-generation lentiviral packaging system: On the basis of the first-generation packaging system, it is safer to remove the auxiliary genes vif, vpr, vpu, and nef that promote virus proliferation and infection.


(3) Third-generation lentiviral packaging system: In order to prevent random recombination from forming a replication-competent provirus, the tat gene was removed from the third-generation packaging system, and the rev gene required for replication was provided in trans. U3 of the 5’LTR was deleted, and the strong promoter CMV was chimeric to initiate transcription. Deleted U3 of the 3′ LTR, including the TATA box and transcription factor binding site. The safety of the system is further improved, but the virus titer will be reduced.


In order to improve the expression and transduction efficiency of lentiviral vectors, modifications can be made.

①Adding transcriptional regulatory elements, such as the central polypurine tract (cppt) and matrix attachment region (MAR), to the cis-expression vector can enhance viral transduction.

②Incorporating the scaffold attachment region of β-interferon gene into the vector design can improve the expression of vector transgene in resting T cells.

③ Using woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) as the post-transcriptional regulatory element of ORF 3’LTR can significantly improve transgene expression.


To achieve tissue or cell type specificity, transgene expression can be driven by the use of tissue specific promoters.

① Use a tetracycline-inducible promoter to regulate the expression of the vector.

② Post-transcriptional regulation of transgene expression by endogenous miRNAs can reduce the immune response to transgene and improve cell-specific targeting.

③ Pseudotyped envelope favors the desired cell or tissue type for successful transduction.


3. Production process of lentiviral vector

The lentiviral vector production process for clinical applications can be divided into upstream production and downstream purification procedures according to GMP production practices.

Upstream production process:

① The virus production of the third-generation vector utilizes transient transfection of the four-plasmid system, and the HEK293 or HEK293T cell line is routinely used. There may be some differences in virus titers between different batches.

② Use a production cell line that stably contains functional lentiviral helper genes and packaging genes. The tetracycline-inducible system was used to control the expression of VSV-G and gag-pol, and the VSV-G pseudotype enhanced the stability of viral particles during vector packaging. Pseudotyped envelopes with less toxicity, such as cocal and RD114-TR, were also developed. The use of stable cell lines has advantages over transient transfection methods in terms of cost, reproducibility, and scalability.

③ Suspension cell culture production can be used for large-scale lentiviral production for clinical use. Compared with adherent cells, serum-free biological contaminants are reduced due to the use of serum-free medium for its culture.


Downstream purification process: centrifugation, filtration steps to remove cellular debris. Various chromatographic techniques can purify viral particles. Buffer exchange and viral particle concentration determination can be performed by ultracentrifugation and tangential flow filtration. Purification strategies for different pseudotyped viruses need to be optimized.


4. Clinical application of lentiviral vectors

In recent years, lentiviruses have been applied in vitro in the form of chimeric antigen receptor (CAR) T cells for refractory hematological malignancies.

In a clinical trial evaluating the safety of 30 pediatric and adult patients with relapsed and refractory acute lymphoblastic leukemia (ALL), the patients’ own T cells were transduced with a transgene-carrying lentiviral vector to express CD19-binding CAR. Patients who received CAR-T cell infusion therapy had a remission rate of up to 90%.

The drug Kymriah is the first CAR-T cell drug approved by the FDA for the treatment of childhood B-cell leukemia. Since then, Yescarta has used gamma retrovirus to stably deliver CAR and is the second CAR-T cell drug approved by the FDA for the treatment of refractory B-cell lymphoma.


How do Viral Vectors-Lentivirus play the role in Gene Therapy?


Many successful clinical trials have shown that lentiviral vectors are safer than other retroviruses.

To date, there are more than 12 completed clinical trials using lentiviral vectors to treat a range of diseases, including metabolic disorders, cancer, immune disorders and rare congenital diseases.


Among them, the in vitro gene therapy strategy of lentivirus for the treatment of genetic diseases has a number of Phase I/II or Phase II/III combined trials in progress:

(1) β-thalassemia: The patient carries a mutation of the HBB globulin gene. After injecting autologous CD34+ cells to transduce the lentiviral vector encoding the human βA-T87Q gene, no significant toxic effects were found during follow-up, and it has been used for the treatment of βA-T87Q. Multinational Phase III clinical trials for thalassemia (NCT03207009 and NCT02906202) and sickle cell disease (NCT04293185).


(2) Cerebral adrenoleukodystrophy (ALD): It is an X-linked recessive genetic disease. If there is no transgenic hematopoietic stem cell transplantation (HSCT), the prognosis is very poor. After patients were injected with lentiviral vector-transduced ABCD1 gene hematopoietic stem cells, 90% overcame disease dysfunction without adverse toxic effects.


(3) Severe combined immunodeficiency (ADA-scid) caused by adenosine deaminase deficiency: The patient was perfused with autologous CD34+ hematopoietic stem cells transduced with ADA gene by lentiviral vector, and survived after two years of treatment. Its drug OTL-101 entered a Phase III clinical trial (NCT04140539) for the treatment of ADA-SCID.


Other lentiviral vectors are also under continuous development, including new vaccines against COVID-19. In two recently initiated clinical trials, lentiviral vectors were used to express synthetic viral minigenes and immunomodulatory genes to design engineered APCs to activate the immune system against COVID-19 (NCT04299724).


5. The future and challenges of lentiviral vectors

Lentiviral vectors are the first choice for transgenic vectors, as compared with gamma retroviral vectors, they have lower genotoxicity such as insertion mutation.

Recently, however, it has been reported that adjacent genes can be activated even with third-generation lentiviral vectors, and lentiviral vectors can cause abnormal splicing of transcripts.

These challenges require further development and retrofit to reduce risk.


Develop safer next-generation lentiviral vectors, which will have more applications in the future:

(1) Transduction of non-dividing cells such as dendritic cells to induce B- and T-cell-mediated immunity against infectious diseases.

(2) Non-integrating lentiviral vectors reduce insertional mutations, can be transiently expressed in dividing cells, evade immune responses, and facilitate the use of CRISPR-Cas systems in therapeutic gene editing.

(3) Integrase-deficient lentiviral vectors have been used as a vaccine platform to provide vaccination against influenza and malaria antigens.









Bulcha JT , Wang Y , Ma H , et al. Viral vector platforms within the gene therapy landscape[J]. Signal Transduction and Targeted Therap.

How do Viral Vectors-Lentivirus play the role in Gene Therapy?

(source:internet, reference only)

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