Exploring the Building Blocks of Vector Design

rAAV vectors are used in approved gene therapies and are being utilized for hemophilia gene therapies currently under investigation.^3,9 Several unique characteristics of AAV make it amenable as a platform for the in vivo delivery of gene therapies,⁹ including:

• Broad tropism (the ability of a virus to infect a particular type of cell in the body) via various serotypes^6,9
• Low immunogenicity^6,9
• Ease of production⁹
• Non-pathogenic in the absence of a helper virus^6,10
• Defective replication, so therefore does not reproduce in the body¹⁰

Wild-type AAV is a single-stranded parvovirus requiring co-infection with a helper virus to facilitate productive infection. AAV comprises a protein shell that surrounds and protects the single-stranded DNA of approximately 4.8 kb.¹¹ This DNA includes open reading frames (ORFs) encoding a promoter, replication and capsid proteins (via the rep and cap genes), flanked by two inverted terminal repeat (ITR) elements.¹¹

rAAV differs from wild-type AAV in that it lacks the viral rep and cap genes.¹¹ Instead, the AAV expression cassette may include gene regulatory elements such as the promoter, the transgene and a transcription terminator (polyadenylation A tail [Poly(A)]).⁸ rAAV is essentially a protein-based nanoparticle that has been engineered to cross the cell membrane and deliver the transgene to the cell nucleus.¹¹ Only the ITRs remain from the wild-type AAV, and are necessary for the proper packaging of the expression cassette into the rAAV.^8,9 The rep and cap sequences are provided separately during production to prevent the generation of wild-type AAV. In the absence of the rep gene within rAAV, the ITR-flanked transgenes can form circular episomal DNA in the nucleus¹¹ (i.e. the rAAV vectors do not have the ability to integrate site-specifically into chromosome 19).¹²

When developing rAAV vectors for gene transfer, there are several aspects to consider: packaging limitation, seropositivity, immune response and transduction efficiency.¹¹

Packaging limitations
The packaging capacity of rAAV is ~5 kb, including the essential viral ITRs.¹¹ This limits the size of the transgene coding sequence that can be packaged within the expression cassette. To find out more about this, click here to access the section on Optimizing Transgene Expression.

Seropositivity
Wild-type AAV, in the absence of a helper virus, does not itself cause infection in humans; however, natural exposure to AAV can result in the formation of neutralizing antibodies against various AAV serotypes.¹⁴ This pre-existing immunity against AAV serotypes may impact the efficiency and limit the delivery of rAAV-based gene therapy.¹⁰

Immune responses
Vector components, including the rAAV capsid and the transgene, may be seen as ‘foreign’ to the immune system, potentially resulting in an immune response leading to destruction of the transduced cells.¹¹ Experience with rAAV8 indicates that the response to rAAV can be long-lasting¹⁵ and neutralizing antibodies against a specific vector type following gene therapy (e.g. anti-rAAV2 antibodies developed in response to treatment with rAAV2 vectors) may preclude further treatment with the same vector.¹⁶ A further challenge is that AAV capsid sequence conservation across multiple serotypes may result in cross-reactivity of neutralizing antibodies over a range of serotypes.^10,17 To learn more about the immune responses associated with gene therapy, click here to access the section on Gene Therapy and the Immune System.

AAV tropism
AAV serotypes exhibit preferential tropism for different tissues meaning that transduction efficacy of specific target tissues varies between serotypes.¹⁶ For example, AAV4 is liver-, lung-, muscle-, eye-, and central nervous system-tropic, with strong tropism for the eye, muscle and the central nervous system.¹⁶ In contrast, AAV6 is liver-, lung- and eye-tropic.¹⁶ An immune response may also interfere with successful transduction of target cells.⁶

To optimize rAAV gene transfer and overcome the limitations of this platform, the rAAV capsid can be engineered to enhance transduction, increase tropism and evade the host immune response.¹¹ There are currently four main approaches used to engineer and optimize the rAAV capsid:

Rational design
Current scientific knowledge (including understanding of the sequence, structure and function) of AAV can be used to further modulate and enhance the performance of the rAAV capsid.^9,18 For example, the introduction of point mutations on tyrosine residues of the vector capsid can decrease proteasomal degradation of the rAAV in the cell and in turn increase transduction efficiency.⁹

Directed evolution
This strategy utilizes genetic diversity and selection processes to allow the accumulation of beneficial mutations that improve the function of the rAAV vector.⁹ For example, through the use of error-prone polymerase chain reaction, it is possible to generate rAAV variants that can potentially evade neutralizing antibodies.⁹

Computer-guided design
This approach employs bioinformatics or machine learning to generate libraries of new AAV capsid variants. These variants can then be screened to identify those with potentially favorable properties such as enhanced transduction abilities.⁹ An example of where bioinformatics has been applied is the construction of potential ancestral AAV capsid libraries to generate alternative capsid variants that can evade the immune response or enhance transduction efficiency.^9,19

Natural discovery
AAV was originally isolated from a cell culture contamination.¹⁹ Vector serotypes isolated from natural sources have been demonstrated to be the most clinically promising vectorized serotypes.¹⁹ For example, AAV9 was isolated from human liver tissue and was demonstrated to bypass the blood–brain barrier, providing an option for gene therapies that can transduce cells within the central nervous system.¹⁹ Additionally, due to the high prevalence of antibodies against various AAV serotypes in the general population, isolation of novel capsids from non-human sources may offer the potential to overcome pre-existing immunity.¹⁹