Lentiviral vs Retroviral – Which Viral Vector Is Right For My Research?

BioInnovatise Viral Vector Team

Updated May 22, 2024

Within the Retroviridae virus family, there are many types of viral vectors used in research and development including Murine Leukemia Virus (MLV), Human T-Cell Leukemia Virus (HTLV), Avian Leukosis Virus (ALV), Mouse Mammary Tumor Virus (MMTV), and of course Lentivirus (HIV-1 Derived).

Lentiviruses and retroviruses are both members of the Retroviridae family and share some common features, such as their replication mechanism involving reverse transcription of their RNA genome into DNA. However, there are many differences in viral structure, application, and capabilities. Our scientists have answered some of your top retroviral vector vs lentiviral vector questions.

What Are The Differences Between Lentivirus and Retrovirus?

Types and Examples:

Lentivirus: A single genus within the Retroviridae family. Examples include Human Immunodeficiency Virus and Simian Immunodeficiency Virus (SIV).
Retrovirus: A broader term that includes several genera besides lentiviruses, such as Alpharetrovirus, Betaretrovirus, and Gammaretrovirus.

Replication Kinetics:

Lentivirus: Capable of infecting both dividing and non-dividing cells. This makes them highly versatile for gene therapy and research applications where targeting non-dividing cells (such as neurons or hematopoietic stem cells) is necessary.
Retrovirus: Typically, retroviruses, like MLV, can only infect dividing cells because their pre-integration complex cannot traverse the nuclear envelope of non-dividing cells. This restricts their use to applications involving rapidly dividing cells.

Genome Structure and Complexity:

Lentivirus: Generally have more complex genomes, encoding additional regulatory proteins (such as Tat and Rev in HIV) that are involved in regulating the viral life cycle and enhancing replication efficiency.
Retrovirus: Simpler genome structures, typically encoding the essential genes gag (structural proteins), pol (enzymes like reverse transcriptase and integrase), and env (envelope proteins), but lacking the additional regulatory proteins found in lentiviruses.

Latency and Pathogenesis:

Lentivirus: Known for establishing long-term latent infections. HIV, for instance, integrates into the host genome and can remain dormant for extended periods, contributing to the chronic nature of the infection.
Retrovirus: While some retroviruses can also establish persistent infections, they generally do not have the same capacity for latency as lentiviruses. The pathology induced by retroviruses varies, with some causing tumors or immunodeficiencies in their hosts.

Integration Site Preferences:

Lentivirus: Integrate relatively randomly into the host genome, but with a slight preference for actively transcribed genes. This makes lentiviral vectors less likely to cause insertional mutagenesis compared to some retroviral vectors.
Retrovirus: Tend to integrate near transcription start sites and regulatory regions, which can increase the risk of insertional mutagenesis and activation of oncogenes.

Biosafety and Vector Design:

Lentivirus: Modern lentiviral vectors are designed with multiple safety features, such as self-inactivating (SIN) LTRs (long terminal repeats) to prevent replication-competent virus formation, and the separation of packaging and vector components onto different plasmids to enhance safety.
Retrovirus: While retroviral vectors also incorporate safety features, the inability to target non-dividing cells and the higher risk of insertional mutagenesis have made them less favorable compared to lentiviral vectors.

Lentiviral vs Retroviral Vector Application Differences in Cell and Gene Therapy:

Lentiviral Vector Applications

Infecting Non-Dividing Cells:
- Hematopoietic Stem Cells (HSCs): Lentiviral vectors are often used for modifying HSCs, which are mostly in a quiescent state. This is necessary for therapies targeting blood disorders, such as beta-thalassemia, sickle cell anemia, and certain immunodeficiencies.
- Neurons: Because neurons are non-dividing, lentiviral vectors are used for gene therapy targeting neurological disorders, such as Parkinson’s disease and spinal muscular atrophy.
- Other Non-Dividing Cells: Lentiviruses are used to deliver genes to a variety of non-dividing cells in tissues like the liver, muscle, and retina for treating diseases like hemophilia, muscular dystrophy, and retinal dystrophies.
Stable Long-Term Expression:
- Chronic Conditions: Lentiviral vectors are suitable for therapies requiring long-term gene expression, such as metabolic disorders and chronic diseases, where sustained expression of therapeutic genes is necessary.
Ex Vivo Gene Therapy:
- CAR-T Cell Therapy: Lentiviral vectors are used to transduce T cells ex vivo with chimeric antigen receptor (CAR) genes, which are then expanded and reinfused into patients to target cancers like leukemia and lymphoma.
Gene Editing:
- CRISPR-Cas9 Delivery: Lentiviral vectors can deliver CRISPR-Cas9 components to target cells for genome editing applications, including gene knock-outs, gene corrections, and insertions for therapeutic purposes.

Retroviral Vector Applications

Infecting Dividing Cells:
- Rapidly Dividing Cells: Retroviral vectors are effective for gene delivery into rapidly dividing cells, such as T cells and certain cancer cells. They have been used in some early gene therapy trials for cancers and inherited immunodeficiencies.
- Ex Vivo Expansion: Retroviral vectors are often used for ex vivo gene therapy where cells are taken from the patient, modified, expanded in culture, and then reinfused. This has been applied in treating conditions like Severe Combined Immunodeficiency (SCID).
Applications Requiring High Integration Efficiency:
1. Oncogenic Studies: Retroviruses’ tendency to integrate near promoter regions can be exploited in research to study oncogene activation and cancer development.
2. Insertional Mutagenesis Research: Due to their integration patterns, retroviral vectors are used in research to understand the effects of insertional mutagenesis, which can provide insights into gene regulation and the development of safer vectors.
Short-Term Expression:
- Transient Therapies: For applications where short-term expression is adequate or desired, retroviral vectors can be utilized, especially in contexts where the cells are expected to divide and dilute out the transgene over time.
Simpler Vector Production:
- Cost and Simplicity: Retroviral vector production can be simpler and more cost-effective compared to lentiviral vectors, making them suitable for certain large-scale or high-throughput applications in basic research and some clinical studies.

Lentiviral Vector Manufacturing and Retroviral Vector Manufacturing Similarities and Differences:

Common Steps in Manufacturing

Plasmid Preparation:
- Plasmids encoding the vector components (transfer vector, packaging plasmids, and envelope plasmid) are prepared and purified.
Transfection:
- Packaging cell lines (such as HEK293T) are transfected with the necessary plasmids using transfection reagents such as calcium phosphate, PEI, or lipofectamine.
Virus Production:
- After transfection, the packaging cells produce and release viral particles into the culture medium over a period of 48-72 hours. Note: Every transfection protocol has differences depending on specific experimental requirements such as the lentiviral system being used and target cells for transduction.
Harvesting:
- The culture medium containing the viral particles is collected.
Purification:
- The viral particles are concentrated and purified, typically using methods such as ultracentrifugation, tangential flow filtration (TFF), or chromatography.
- Our lab purifies lentiviral and retroviral vector productions using ultracentrifugation to increase titer yield.

Specific Differences in Manufacturing

Lentivirus Packaging

Packaging System:
- Lentiviral vectors often require a more complex packaging system, typically involving a three- or four-plasmid system depending on the generation of lentivirus. This includes:
  - The transfer vector plasmid carrying the gene of interest.
  - A plasmid encoding Gag and Pol proteins.
  - A plasmid encoding the Rev protein (specific to lentiviruses).
  - An envelope plasmid (commonly VSV-G for broad tropism).
- Biosafety Features:
  - Lentiviral vectors incorporate safety features such as self-inactivating (SIN) LTRs to prevent replication-competent virus formation and minimize the risk of insertional mutagenesis.
- Transduction of Non-Dividing Cells:
  - The production process must ensure that the viral particles are capable of efficiently transducing non-dividing cells. This requires careful optimization of the envelope protein and other vector components.

Retrovirus Packaging

Packaging System:
- Retroviral vectors typically use a simpler two- or three-plasmid system, which includes:
  - The transfer vector plasmid carrying the gene of interest.
  - A plasmid encoding Gag and Pol proteins.
  - An envelope plasmid (either a specific retroviral envelope or VSV-G for broader tropism).
Integration and Tropism:
- Retroviral vectors are primarily used for transducing dividing cells. The production process is optimized for these applications, focusing on achieving high titers and ensuring stable integration in dividing cells.
Cell Lines:
- Different packaging cell lines may be used depending on the retrovirus. Our lab produces all lentiviral and retroviral productions with a HEK293T cell line unless otherwise noted.

Lentivirus Packaging Generations vs Retrovirus Packaging Generations Similarities and Differences:

Similar to lentiviruses, retroviral vectors also have different generations that reflect advancements in their design and safety features. These generations have been developed to improve the efficiency, safety, and utility of retroviral vectors for gene therapy and research applications.

Lentiviral Vectors: Lentiviral vectors have different generations,, each incorporating improvements in safety and efficiency, similar to retroviral vectors. Lentiviral vectors, especially third-generation vectors, are designed to minimize the risk of RCR formation and have features like SIN LTRs.
Retroviral Vectors: While both types of vectors have evolved to improve safety and functionality, retroviral vectors are generally simpler and primarily used for applications involving dividing cells. Lentiviral vectors, on the other hand, are more complex and can efficiently transduce non-dividing cells, making them suitable for a broader range of applications.

Can The Same Plasmid DNA Construct Be Inserted Into A Lentiviral Vector And A Retroviral Vector?

In theory, yes, the same plasmid DNA construct encoding a gene of interest (GOI) can be inserted into both lentiviral and retroviral vectors. However, important considerations and modifications need to be made due to the differences in these vector systems. If your plasmid DNA construct is not optimized for the specific viral vector you are using, you may experience lower viral titers and poor infection rates.

Here are some of the considerations for each viral vector:

LTR Sequences:
- Retroviral Vectors: Use specific LTR sequences derived from the retrovirus (e.g., MLV). These LTRs are necessary for the integration and expression of the retroviral genome.
- Lentiviral Vectors: Often employ self-inactivating LTRs to reduce the risk of insertional mutagenesis.
Packaging Signal:
- The packaging signal required for the packaging of the viral RNA genome into viral particles is specific to the type of virus. The packaging signal in a retroviral vector is different from that in a lentiviral vector.
Regulatory Elements:
- Lentiviral Vectors: Often include additional regulatory elements like the central polypurine tract and the Rev response element, which are specific to lentiviral replication and nuclear import.
- Retroviral Vectors: Typically do not include these elements, as they are not necessary for retroviral vector function.
Promoter and Enhancer Sequences:
- Both types of vectors can use similar internal promoters, such as CMV, EF-1α, PGK, but the choice of promoter might be influenced by the specific application and cell type being targeted.
Vector Backbone:
- The overall design and backbone of the plasmid may differ to accommodate the specific requirements of lentiviral or retroviral vector production.

If your plasmid DNA construct requires modifications such as molecular cloning for a specific backbone, contact our cloning team.

Ensuring the appropriate viral vector for your research is incredibly important. If you are not sure which viral vector is right for your research and development, contact our viral vector team.

Learn about our quick turnaround lentivirus packaging services and retrovirus packaging services.

Want to learn more about the latest in lentivirus or retrovirus based research? Our colleagues at ScienceDirect (lentivirus and retrovirus) and Genetic Engineering & Biotechnology News (lentivirus and retrovirus) continuously collect and publish the latest information on lentivirus-based research.

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