Welcome to Gene Therapy Science / Vector Design

Gene Therapy Vectors

Hemophilia gene transfer aims to deliver a functional copy of the F8 or F9 gene to the hepatocyte cells in the liver.1 Naked DNA plasmids are rapidly degraded in biological fluids (and therefore unable to reach the target cells), unless delivery vectors are utilized to protect the nucleic acids.2 The functional gene (referred to as a transgene) is packaged into a capsid, resulting in a vector that can deliver the transgene to the nucleus of the target cell, where the cell can then express the therapeutic protein of interest.3,4 The success of gene therapy is therefore dependent on the development of effective vectors that act as vehicles for gene transfer.1

There are two main groups of gene therapy vectors: viral vectors and non-viral vectors. The application of non-viral vectors has been very limited to date due to poor efficiency of gene delivery.5 The success of gene therapy therefore depends on effective vehicles for gene transfer that are based on recombinant viral sequences, but are not viruses themselves.1

There are four main viral vector groups currently under investigation for gene therapy:

Two RNA-based viral vectors–gammaretrovirus and lentivirus (both classified as retroviruses)6,7 Two DNA-based viral vectors–adenovirus and recombinant adeno-associated virus (rAAV).6
Image Vector Genetic Material Packaging Capacity Tropism Vector Genome Forms Select Limitations Select Advantages
Hemo Gene Icon 1 Gamma –
retrovirus 6,7
RNA 6 8 kb 6 Dividing cells only 6 Integrated 6

Only transduces dividing cells and integration into the host genome occurs at random sites 6

Persistent gene transfer in dividing cells 6
Hemo Gene Icon 2 Lentivirus 6 RNA 6 8 kb 6 Broad (dividing and non-dividing cells) 6 Integrated 6 Random integration may cause potential complications 6 Persistent gene transfer in most tissue 6
Hemo Gene Icon 4 Adenovirus 6

Double-stranded DNA 6

Replication defective:   8 kb 6

Helper-dependent: 30 kb 6

Broad (dividing and non-dividing cells) 6 Episomal 6 Capsid mediates a potent inflammatory response 6 Extremely efficient transduction of most tissues 6
Hemo Gene Icon 3 Recombinant adeno-associated virus 6 Single-stranded DNA 6 <5 kb 6 Broad (dividing and non-dividing cells) 6 Predominantly episomal. Random integration events observed with a low frequency (0.1–1% of transduction events) 8 Small packaging capacity 6 Non-inflammatory; non-pathogenic 6

Key properties of the four main vector groups currently under investigation for use in gene therapies. Table developed from Thomas CE, et al. 2003,6 Maetzig T, et al. 20117 and Colella P, et al. 2018.8

Recombiant AAV is Commonly used for Hemophilia Gene Therapy3

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 Versus Recombiant AAV 

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    

Considerations with Recombinant AAV Vectors

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 .
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​​​​
Engineering the Recombinant AAV Capsid to Optimize Gene Therapy

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
Directive 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
Learn More About Optimizing
Transgene Expression
Learn more
1. Lheriteau E, et al. Blood Rev 2015;29(5):321–8. 2. Nobrega C, et al. A Handbook of Gene and Cell Therapy. Springer International Publishing; 2020. 3. Sidonio R, Jr. Blood Rev 2020;100759. doi: 10.1016/j.blre.2020.100759 (Online ahead of print). 4. Kumar SR, et al. Mol Ther Methods Clin Dev 2016;3:16034.
5. Ramamoorth M, Narvekar A. J Clin Diagn Res. 2015;9(1):GE01–6. 6. Thomas CE, et al. Nat Rev Genet 2003;4:346–58. 7. Maetzig T, et al. Viruses 2011;3(6):677–713. 8. Colella P, et al. Mol Ther Meth Clin Dev 2018;8:87–104. 9. Li C, Samulski RJ. Nat Rev 2020;21:255–72. 10. Mingozzi, F, et al. Blood 2013;122(1):23–36. 11. Naso MF, et al. BioDrugs 2017;31(4):317–34. 12. Daya S, Berns KI. Clin Microbiol Rev 2008;21(4):583–93. 13. Pfeifer A, et al. Annu Rev Genomics HumGenet 2001;2:177–211.
14. Verdera HC, et al. Mol Ther 2020;28(3):723–6. 15. Nathwani AC, et al. N Engl J Med 2014;371:1994–2004. 16. Grimm D, Kay MA. Curr Gene Ther 2003;3:281–304. 17. Vandamme C, et al. Hum Gene Ther 2017;28(11):1061–74. 18. Lee JE, et al. Curr Opin Biomed Eng. 2018;7:58–63. 19. Wang D, et al. Nat Rev 2019;18:358–78.
Date of preparation: February 2022. ​​​​​​​

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