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Optimizing Transgene Expression

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Gene therapies for hemophilia are currently being studied to determine their safety and efficacy. No gene therapies for hemophilia have been approved for use.

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Overview of the Recombinant Adeno-Associated Virus (rAAV) Expression Cassette

Once F8 or F9 transgenes are delivered to hepatocyte cells in the liver, transduced cells can potentially express FVIII or FIX proteins at therapeutic levels to replace the missing or nonfunctioning coagulation factor.1,2 In order to control and optimize transgene expression, an understanding of each component of the rAAV transgene expression cassette is key.3,4 The rAAV expression cassette may comprise of five key elements:

The transgene is the nucleic acid sequence encoding the therapeutic gene of interest (F8 for hemophilia A or F9 for hemophilia B).2,3,5 Following transduction, the coding region can be transcribed and subsequently translated using the endogenous transcription and translation machinery to produce functional protein.2,3

A promoter is a sequence of DNA, typically in the five prime (5’) region of the expression cassette, where regulatory elements such as transcription factors bind and initiate transcription of the associated gene.6 In the rAAV expression cassette, the promoter sequence sits upstream of the transgene.3,7

An enhancer element is an upstream regulatory DNA sequence that can augment the activity of a promoter. The enhancer sequence comprises binding sites for regulatory proteins that, when bound, trigger chromatin changes that promote RNA polymerase and transcription factor binding, thus improving the transcriptional ability of the associated promoter.7

Inverted terminal repeats (ITRs)
ITRs are 145–base pair sequences that flank the expression cassette.8 The ITRs are crucial for vector production and transgene expression and are the only genetic elements retained from wild-type AAV.3,8 In the absence of the rep gene, which can be found in the wild-type AAV genome, the ITR-flanked transgenes can form circular episomal DNA in the nucleus. In contrast to wild-type AAV genomes, rAAV vector genomes do not integrate specifically into chromosome 19 in human cells in vitro, and have been shown to mainly remain episomal in animal models in vivo.8

Transcription terminator (polyadenylation tail [Poly(A)])
The Poly(A) signal sequence acts as the transcription terminator, halting transcription once the transgene is transcribed.9 The inclusion of this sequence is also critical for nuclear export, translation, and mRNA stability.4

rAAV Expression Cassette


    For successful transgene expression, the gene of interest must fit within the packaging capacity of the vector to enable transport to the target cells.12,14 For rAAV, this packaging capacity is ≈5.0 kb.12,14 If a transgene exceeds this size, the complementary DNA (cDNA) may need to be altered to overcome this barrier.11 Additionally, transgene expression can potentially be improved through the optimization of the transgene itself (e.g. via codon optimization).3 For hemophilia gene therapy, there are various considerations when selecting and optimizing F8 or F9 transgenes.11

    Hemophilia A – F8 gene size
    FVIII is encoded by the F8 gene, which is ≈7 kb in size. This far exceeds the limited packaging capacity of rAAV, and therefore, the F8 cDNA must be reduced in size in order to be packaged within rAAV.11

    The structure of the F8 gene is well documented and is known to comprise three homologous A domains, two homologous C domains, and a unique B domain that constitutes 38% of the primary cDNA.15 The A and C domains are essential for the procoagulant activity of FVIII; however, deletion of the B domain does not appear to impair protein function.15 While data have suggested that complete deletion of the B domain may impair post-translational trafficking and secretion,2,16 constructs exploring partial deletion of the B domain have been explored.2 Partial deletion of the F8 B domain has been shown to lead to an increase in mRNA levels and an increase in secreted FVIII protein compared with wild-type F8.2 Recombinant B-domain-deleted (BDD)–FVIII is widely used as a replacement factor for the treatment of people with hemophilia A,2 and adopting a similar approach of using BDD-F8 cDNA for gene therapy can address the packaging restrictions of rAAV.11

    Hemophilia B – F9 gain-of-function mutation
    FIX is encoded by the F9 gene, which is 1.6 kb in size.11 This small gene can be packaged into the rAAV expression cassette without any modifications to its size.2,11

    Optimization of F9 for gene therapy has focused primarily on increasing gene expression following delivery of the transgene to the target cells. This can be achieved by incorporating a gain-of-function point mutation in the F9 gene. The naturally occurring gain-of-function point mutation (R338L), named F9-Padua, has been shown to lead to an increase in FIX activity versus wild-type FIX.16,17 Use of F9-Padua cDNA is being investigated for hemophilia B gene therapies.16

    rAAV Figure 2

    Codon optimization of F8 and F9 genes
    Transgene codon optimization is an approach used to potentially maximize the expression and therapeutic potential of a particular transgene.21 Most amino acids are encoded by more than one codon, with different organisms and cell types showing bias towards certain codons.22 The goal of codon optimization is to match codon usage in the transgene with the abundance of transfer RNA for each codon in a particular cell type21 through the use of synonymous codon substitutions.22 This, in turn, may increase the rate and efficiency of translation by using more abundant codons.22

    Expression of the F8 and F9 transgenes can be further improved through codon optimization, which is now standard practice when expression cassettes and vectors are being developed for clinical studies.3 Several different codon-optimized F8 transgenes have been demonstrated to improve expression levels of FVIII.2,16 For F9 transgenes, codon optimization typically results in an increase in expression.16 However, data have shown that codon optimization of F9 may impact protein conformation and lead to different translation kinetics compared with wild-type FIX.22 These changes can be unpredictable and require further investigation.22


    In addition to optimizing the transgene, the expression cassette can be further enhanced through consideration of the promoter and enhancer elements.4

    Promoters are essential for transgene expression,10 and can be ubiquitous or tissue-specific.4

    Ubiquitous promoters, such as elongation factor 1α-subunit and immediate-early cytomegalovirus (CMV), can be used to promote expression across many tissue types. The level of expression can vary between tissues and cell types.4

    Tissue- or cell-specific promoters allow for greater control of transgene expression.4 Hemophilia gene therapy aims to establish F8 or F9 transgene expression in liver hepatocyte cells.11 By using promoter sequences from proteins naturally synthesized in hepatocytes, such as α1 antitrypsin or thyroxine-binding globulin, the expression of F8 and F9 can be targeted to the liver with minimal expression in other tissues, therefore reducing the likelihood of a transgene-induced immune response.3,4,6 Additionally, natural endogenous promoters of the therapeutic gene offer the possibility of directing expression in relevant cells, at the appropriate level and time.23

    Modification of strong, large promoters is also under investigation, with small cis elements possibly replacing long promoter sequences.3

    Enhancers support the activity of promoters by providing binding sites for regulatory proteins.7 An in vitro study of enhancer / promoter combinations for optimal FVIII expression in nonviral vectors found that the combination of chimera enhancer HCR-1 / HCR-2 and CMV promoter led to an increase in factor expression compared with only the promoter alone.24


    Introns can also be used to optimize transgene expression. In a study optimizing rAAV vectors for liver-directed gene therapy, it was shown that placing an intron between the promoter and transgene resulted in higher transgene expression than if no intron was included, with this effect being most apparent with the intron from minute virus of mice (MVM, from the parvovirus family of viruses).4,25,26


    An additional consideration relates to the biology of the single-stranded AAV-delivered transgenes. After delivery to the nucleus, the single-stranded transgene needs to be converted into a double-stranded transgene before expression can occur.27 This rate-limiting step to gene expression can be bypassed using self-complementary (sc) DNA vectors, referred to as scAAV.27 scAAV contains both the coding and complementary sequence of the transgene expression cassette.27

    scAAV vectors have been demonstrated to be more potent than single-stranded vectors and are currently being investigated for the delivery of F9 in some clinical trials.2,28 Because of the reduced packaging capacity of scAAV, this approach is not currently being investigated for the delivery of F8.2,27


1. Crudele JM, et al. Blood 2015;125(10):1553–61. 2. Lheriteau E, et al. Blood Rev 2015;29(5):321–8. 3. Li C, Samulski RJ. Nat Rev Genet 2020;21(4):255–72. 4. Powel SK, et al. Discov Med 2015;19(102):49–57. 5. George LA. Blood Adv 2017;1(26):2591–9. 6. Ohmori T. Int J Hematol 2020;111:31–41. 7. Nature scitable, gene expression. (Accessed April 2021). 8. Daya S, Berns KI. Clin Microbiol Rev 2008;21(4):583–93. 9. Tran DP, et al. Mol Cell Biol 2001;21(21):7495–508. 10. Zheng C, Baum BJ. Method Mol Biol 2008;434:205–19. 11. Doshi BS, Arruda VR. Ther Adv Hematol 2018;9:273–93. 12. Thomas CE, et al. Nat Rev Genet 2003;4(5):346–58. 13. Pfeifer A, et al. Annu Rev Genomics Hum Genet 2001;2:177–211. 14. Chamberlain K, et al. Hum Gene Ther Methods 2016;27(1):1–12. 15. Saenko EL, et al. J Thromb Haemost 2003;1(5):922–30. 16. VandenDriessche T, Chuah MK. Hum Gene Ther 2017;28(11):1013–23. 17. Simioni P, et al. N Engl J Med 2009;22(361):1671–5. 18. High KA, Roncaralo MG. N Engl J Med 2019;381:455–64. 19. Nguyen GN, et al. J Throm Haem 2016;15:110–21. 20. Samelson-Jones BJ, et al. JCI Insight 2019;4(14):e128683. 21. Kattenhorn LM, et al. Hum Gene Ther 2016;27(12):947–61. 22. Alexaki A, et al. Sci Rep 2019;9:15449. 23. Le Bec C, Douar AM. Gene Therapy 2006;13:805–13. 24. Picanço-Castro V, et al. Genet Mol Res 2008;7(2):314–25. 25. Wu Z, et al. Mol Ther 2008;16(2):280–9. 26. Agbandje-McKenna M, et al. Structure 1998;6(11):1369–81. 27. Naso MF, et al. BioDrugs 2017;31(4):317–34. 28. Batty P, Lillicrap D. Hum Mol Genet 2019;28(R1):R95–R101.

Date of preparation: April 2021

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