AAV vs Lentivirus – Which Viral Vector Is Right For My Research?

BioInnovatise Viral Vector Team

Updated September 23, 2024

Many therapeutics, vaccines, and treatments in discovery phase are using AAV (adeno-associated virus) and lentivirus platforms, and several candidates have reached clinical trials and commercialization.

Researchers need to understand the differences between these two mainstream viral vectors when it comes to delivering the specific genetic payload in order to infect host cells. There are differences in viral structure, applications, and capabilities. Our viral vector scientists have answered some of your top AAV vs lentivirus questions.

AAV vs Lentivirus: What Are The Viral Vector Structural Differences?

Most researchers are familiar with the basic structure of the AAV vector or lentiviral vector, however the specific cargo capacity and structural differences are important for specific research applications.

Capsid Structure:

Adeno-Asscoaited Virus:

AAV is a non-enveloped virus, meaning it lacks a lipid membrane surrounding its capsid.
- Icosahedral Capsid: AAV has a symmetric, icosahedral capsid structure made of protein subunits, specifically three viral proteins (VP1, VP2, VP3) arranged in a precise geometric pattern.
- Spike Surface Capsid Proteins: The capsid plays a critical role in determining the tissue tropism and immune response. Different serotypes of AAV have distinct capsid structures that allow them to target different tissues. Serotypes are discussed further below.

Lentivirus:

Lentiviruses, which are a subset of retroviruses, are enveloped viruses. They have a lipid bilayer derived from the host cell membrane that surrounds the viral core.
- Spherical or Cone-shaped Core: Inside the envelope, lentiviruses have a core that is often described as conical or bullet-shaped, which houses the viral genome and essential enzymes like reverse transcriptase and integrase.
- Glycoproteins in Envelope: The viral envelope contains glycoproteins (e.g., the HIV-derived envelope protein gp120), which are responsible for receptor binding and entry into the host cell.

Cargo Capacity:

Lentiviral vectors can package up to about 8–12 kb of foreign DNA. This larger size capacity makes lentiviruses suitable for delivering more complex or larger therapeutic genes.
AAV vectors have a smaller packaging capacity of around 4.7 kb. This is a significant limitation when it comes to delivering larger genes or additional regulatory sequences.

Depending on the size of the genetic payload, AAV may not be a feasible viral vector for your research application. This is the largest reason why researchers choose lentivirus vs adeno associated virus.

Genomic Architecture Differences:

Lentiviral plasmids use Long Terminal Repeats (LTRs) and packaging signals (ψ) that are required for reverse transcription and integration into the host genome. AAV plasmids do not require LTRs or these packaging signals, making a direct swap impossible without modification.
AAV vectors require specific Inverted Terminal Repeats (ITRs) flanking the gene of interest. These ITRs are essential for packaging the therapeutic gene into the AAV capsid and facilitating its transduction into target cells. Lentiviral plasmids do not have these ITRs, so a plasmid designed for AAV cannot be directly used for lentivirus without removing the ITRs.

Plasmid DNA requirements and differences for AAV packaging and lentivirus packaging are summarized further below.

Host Cell Integration:

Lentivirus integrates its DNA into the host cell genome, allowing for long-term expression in dividing cells. Therefore, lentivirus packaging plasmids are designed with regulatory elements, such as LTRs that support this integration process.
AAV, in contrast, remains largely episomal (non-integrated) in the host cell nucleus and primarily targets non-dividing cells. AAV packaging plasmids are optimized for episomal persistence and long-term expression without integration into the genome.

The long term expression and host cell integration differences are one of the biggest reasons researchers choose AAV vs lentivirus.

Based On Cargo Capacity Differences, What Are The Downstream Considerations In Gene Therapy Application?

Choosing between AAV vs lentivirus for gene therapy is highly dependent on the cargo size requirements and the downstream considerations such as immune response, gene expression stability, tissue targeting, and safety concerns. We have broken down each of these considerations to compare lentivirus vs AAV in gene therapy.

Gene Selection:

Due to AAV’s smaller packaging capacity, only small genes can be used to successfully infect host cells. This limitation necessitates careful selection or modification of the therapeutic gene, potentially affecting its full functionality. Molecular cloning a plasmid DNA construct may shorten a construct’s length enough to get it packaged into an AAV.
With a larger capacity, lentiviral vectors can accommodate more complex or larger genes without significant alterations during lentivirus packaging or lentiviral manufacturing. This clearly allows for more flexibility in gene selection.

Regulatory Elements and Promoter Choices:

The limited cargo size for AAV restricts the inclusion of regulatory elements such as promoters, enhancers, and insulators. Small and highly efficient promoters must be chosen to fit within the size constraints, which may affect the gene’s expression levels and tissue specificity.
Lentiviral vectors can incorporate larger and more sophisticated regulatory elements, allowing for more precise control of gene expression, tissue targeting, and reduced risk of off-target effects.

Gene Editing and CRISPR-Cas9 Utilization:

AAV’s smaller size limit restricts the use of more complex therapeutic strategies, such as those involving CRISPR-Cas9 vectors, CAR genes, multiple genes, or large cDNAs.
Lentiviral vectors can incorporate larger and more sophisticated regulatory elements, allowing for more precise control of gene expression, tissue targeting, and reduced risk of off-target effects.

Immune Response and Biosafety:

Smaller AAV vectors are generally considered to have a low immunogenic profile, but the limited size means fewer opportunities to include safety elements like immune-modulating sequences. There is a risk of higher immune response due to pre-existing antibodies against specific AAV serotypes.
Lentiviral vectors allow the inclusion of safety switches and immune evasion strategies, which can help reduce immune responses. However, lentiviral vectors do integrate into the genome, raising concerns about insertional mutagenesis and the risk of disrupting oncogenes or tumor suppressor genes.

Tissue and Disease Targeting / Serotype Options:

AAV is often limited in its ability to deliver therapeutic genes to a wide variety of tissues unless small genes or split systems are used. However, certain AAV serotypes have been optimized for specific tissues such as AAV8 for neuroscience research.
The larger cargo limit of lentiviral vectors enable the delivery of more sophisticated therapies to various tissues, making them versatile for treating a broader range of diseases.

Therapeutic Efficacy and Longevity:

AAV’s limited capacity may impact the ability to include elements that enhance therapeutic efficacy, such as targeting specific cell types, or immune response mitigation.
Lentiviral vectors’ larger cargo capacity allows the vector to include various elements that can improve therapeutic efficacy, such as enhancers, tissue-specific promoters, or additional transgenes. Integration into the host genome also allows for sustained gene expression in both dividing and non-dividing cells, but there is a risk of insertional mutagenesis as previously mentioned.

AAV vs Lentivirus: Can The Same Plasmid DNA Construct Be Used?

The same plasmid DNA construct cannot typically be inserted directly into both a lentiviral vector and an AAV vector without significant modifications because of the above mentioned reasons. While both vectors can carry therapeutic genes, they have different structural, regulatory, vector cargo capacity limits, and functional requirements that necessitate specific plasmid design considerations.

If you need your plasmid DNA construct edited to fit a specific viral vector, contact our molecular cloning team.

What Are The Similarities And Differences Between AAV and Lentivirus Packaging?

Similarity: Packaging Plasmids

Both AAV packaging and lentivirus packaging involve transfection of helper plasmids into the cell line.
For each production, multiple plasmids are used to provide the necessary viral proteins, genome packaging signals, and replication functions. The plasmids generally include a transfer plasmid carrying the therapeutic gene and packaging plasmid(s) encoding viral proteins essential for assembling the viral vector.

Similarity: Transient Transfection of Producer Cells

For both vectors, production often occurs via transient transfection of producer cells, using chemical transfection agents or electroporation. Note: Each transfection protocol differs depending on the end research application.
Transient transfection leads to temporary expression of viral proteins and packaging of the therapeutic gene into viral particles.

The above diagram illustrates the production process for lentivirus packaging at BioInnovatise.

Similarity: Cell-based Production Systems

Both AAV and lentivirus production systems rely on cell cultures such as adherent or suspension cell systems to generate viral particles. In large-scale production (read more about lentiviral vector manufacturing), suspension cell culture systems in bioreactors are preferred to increase yield.

Similarity: Purification Methods

Both viral vectors undergo similar purification steps, including filtration and chromatography to separate the viral particles from impurities. Our team uses ultracentrifugation to purify viral vector productions.

Difference: Helper Plasmid(s)

AAV requires helper virus components for its replication and packaging while lentivirus does not. These helper plasmid components for AAV are used in the transfection process.

Difference: Number of Plasmids Used During Co-Transfection

AAV packaging requires a three-plasmid system: one for the AAV genome (therapeutic gene), one for AAV rep/cap genes (replication and capsid proteins), and one for the adenoviral helper genes (to support AAV replication)
Depending on the lentivirus packaging generation, lentivirus packaging can require 3 or 4 plasmids to increase safety. Read more about lentivirus packaging generations.
- Second generation lentivirus packaging uses 3 plasmids: one for the transfer vector (containing the gene of interest), one packaging plasmid (providing essential viral proteins), and one
  envelope plasmid (encoding the viral envelope protein).
- Third generation lentivirus packaging includes all the second generation packaging plasmids but separates the gag/pol and rev plasmid vectors to reduce reduce the likelihood of recombination events.

Viral Particle Assembly

AAV assembly takes place in the nucleus of the producer cells, where the single-stranded AAV genome is packaged into capsids. The viral particles are then released upon cell lysis.
Lentiviral vector assembly occurs in the cytoplasm, where RNA is reverse-transcribed, packaged, and then enveloped by the cell membrane as it buds off from the cell, without requiring cell lysis.

Yield and Production Efficiency

AAV production is generally challenging due to its smaller genome and complex assembly. It has lower yields compared to lentivirus, and the efficiency of packaging declines as the genome approaches its packaging capacity.
Lentivirus tends to have higher production yields and better efficiency in packaging, even when the genome approaches its packaging capacity.

Purification Complexity

AAV often requires more stringent purification methods due to the presence of incomplete or empty capsids. Empty capsids can make up a significant proportion of the total viral particles produced, necessitating additional steps to remove these.
Lentivirus typically produces fewer empty particles, making purification somewhat simpler, although contaminants like host cell proteins and DNA must still be removed via ultracentrifugation.

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.

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

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