Virus-derived vector systems have been used as gene delivery tools in gene and cell therapy for many years. There are many types of viral vectors available, of which lentivirus (LV), gamma-retrovirus (GRV), adenovirus (AV), and adeno-associated virus (AAV) are the most commonly used in gene and cell therapy. Gamma-retroviral vectors (GRVV) and lentiviral vectors (LVV), derived from mouse leukemia virus and HIV, respectively, are the two most commonly used retroviruses. The main factors to consider when selecting a viral vector system for a specific application include the amount of therapeutic GOI (gene of interest) loaded (insert size), immunogenicity, cell tropism and efficiency of uptake by target cells, duration of gene expression, and simplicity of large-scale production.

Persistence of therapeutic gene expression is a key consideration in selecting a viral vector for the application requirements. Viral vectors can be divided into two categories: integrating and non-integrating. Both gamma-retroviral and lentiviral vectors are retroviruses with an RNA genome that integrates in dividing cells and usually results in sustained expression of the GOI, whereas AAV and adenovirus are non-integrating DNA viruses that result in transient expression in dividing cells. Another advantage of gamma-retroviral and lentiviral vectors is their gene insert capacity of approximately 9 kb.

Integrative Viral Vectors: Retroviruses

Retroviruses are enveloped RNA viruses that contain two copies of a single-stranded RNA genome. The envelope plays a very important role in determining host cell (HC) specificity. Envelope proteins facilitate cell entry through direct membrane fusion or receptor-mediated endocytosis facilitated by binding of envelope glycoproteins to cognate receptors on host target cells.

Retroviral vectors are a popular choice for gene therapy and cell therapy because they can achieve long-term stable gene expression by integrating into the host cell genome. However, integration also has risks, which may lead to insertional mutagenesis, resulting in upregulation of proto-oncogenes and malignant transformation of host cells. Gamma-retroviral vectors (GRVV) tend to integrate in close to gene regulatory regions, such as transcription start sites, which poses a higher risk of genotoxicity than lentiviral vectors that tend to integrate into the gene body. Another major difference between gamma-retroviral vectors and lentiviral vectors is that gamma-retroviral vectors preferentially transduce dividing cells and cannot transduce non-dividing cells, while lentiviral vectors can transduce both dividing and non-dividing cells. Gamma-retroviral vectors have a simple genome structure consisting of the following protein-coding genes: gag, pol, and env. Gag encodes the capsid protein, Pol encodes the viral enzymes (reverse transcriptase, integrase, and protease), and Env encodes the envelope protein. Lentiviral vectors have a more complex genome. In addition to gag, pol, and env, the HIV genome encodes 6 additional proteins: 2 regulatory proteins Rev and Tat and 4 accessory proteins Vpr, Vpu, Vif, and Nef.

To mitigate the adverse effects associated with the pathogenicity of wild-type retroviruses and other potentially harmful effects, retroviral vectors have been made safer by reducing the viral genome to only the essential genetic elements required for efficient packaging into viral particles. In addition to deleting unnecessary genetic elements to make viral vectors safer, the viral genome has been segmented and the packaging genome is provided in trans. The latter is delivered via packaging system cell lines used to generate stable vector production cell lines or different plasmids for transient production of viral vectors. This reduces the chance of recombination and renders the viral vectors replication-incompetent, thereby improving safety.

The third generation of lentiviral vectors consists of a 4-plasmid system, in which the four auxiliary proteins Vpr, Vpu, Vif and Nef are removed, and the vector genome is reduced to only encoding 3 of the 9 HIV proteins, making it safe for use as a gene delivery vector. In addition, the 3 HIV genes and the envelope (from different species) are split into:

• Delivery vector containing LTR, packaging signal Ψ, Rev response element (RRE) and PPT from the viral genome and GOI
• Rev regulatory element
• Gag/Pol packaging plasmid, and
• Envelope plasmid

Currently, the fourth generation of lentiviral vector systems have been developed, which further split the viral genome into a five-plasmid system. However, this approach requires more plasmids and may reduce the efficiency of transfection. In addition, the homology between the transfer plasmid and the packaging plasmid was further reduced by codon optimization, but this led to a decrease in viral titer. Berkhout et al. redesigned the transfer plasmid to place the viral cis-acting elements downstream of the 3' LTR so that these elements would not be integrated into the host cell genome, making the vector system safer. However, the fourth-generation lentiviral vector system requires more development work to make it as efficient a gene delivery tool as the third-generation system.

The first approved gene therapy vector was the Moloney murine leukemia virus (MLV) γ-retroviral vector for SCID-X1 (human severe combined immunodeficiency X-linked) patients. Of the five CAR-T products currently approved by the FDA, two use γ-retroviral vectors and three use lentiviral vectors. Lentiviral and γ-retroviral vectors are the most attractive gene delivery systems due to their low immunogenicity and high transduction efficiency.

Retroviral Vector Production Processes

There are two main production platforms for gamma-retroviral and lentiviral vectors: transient processes, which involve transfection of plasmids (multiple or single) into appropriate cell lines, and stable processes, which involve the preparation of stable cell lines that produce viral vectors using packaging cell lines that express packaging and envelope genes.

In the early stages, i.e., research and development, transient processes are preferred because they are faster and easier to implement. Using transient platforms, the functionality of multiple viral constructs can be tested simultaneously before the final construct is selected. Once the vector construct for the project is determined, the generation of stable viral vector production cell lines using the selected vector construct can begin. It takes about a year to prepare a cGMP-compliant master cell bank (MCB), but a stable vector production cell line will make the production of vectors for clinical and commercial purposes much easier.

Typically, small-scale production is performed in the early stages of research and development, and process optimization studies are performed at this stage to improve the yield and quality of the vector. At the same time, research-grade materials and reagents that do not have very strict quality requirements can be used at this stage. As projects transition from the R&D stage to the preclinical, clinical and commercialization stages, not only will the scale of production increase, but GMP-grade reagents, cell lines and plasmids will also be required to meet the stringent quality and regulatory requirements issued by regulatory agencies. Therefore, costs will increase, which means that in order to achieve cost-effective viral vector production, cost factors must be taken into account when determining the best production technology.

The production process of transient platforms and stable platforms can be divided into two stages: upstream and downstream. The upstream process produces viral vectors with a suitable cell line and obtains a large amount of viral supernatant. The downstream process includes purification steps for removing process and product-related impurities, as well as concentration steps to obtain purified, effective viral vectors to achieve the effectiveness of gene delivery.

Lentiviral Vector Manufacturing

Creative Biogene's manufacturing infrastructure features GMP-compliant dedicated cell bank establishment areas, aseptic isolators for final product filling, and optimized processes that significantly reduce production and testing costs. Both the adherent and suspension platforms are supported by comprehensive documentation and traceability systems that fully satisfy regulatory requirements for clinical and commercial applications, ensuring a seamless transition from development to market approval.

We offer two robust lentiviral production systems with FDA DMF registration and clear IP traceability: