Translational Vectorology

Recombinant viral vectors derived from Adeno-Associated Virus type 2 (AAV2) are powerful tools for both gene addition and gene repair. The parental virus is non-pathogenic and requires co-infection with helper virus for productive infection. The single-stranded DNA genome consists of two inverted terminal repeat (ITR) sequences (145bp) flanking open reading frames (ORFs) encoding the viral Rep and Cap proteins. The ITRs contain the cis-acting viral sequences required for genome replication and encapsidation [4]. This structure allows the generation of recombinant AAV vectors that retain only the ITR sequences. Vector stocks can be generated at high titre by supplying the viral gene products in trans. A critical advance in the AAV field has been the discovery that the AAV2 genome can be cross-packaged into the capsids of other AAV serotypes (pseudo-serotyping) [5] and with engineered capsid variants [6]. This alters vector tropism, immuno-biology, the kinetics of transgene expression, intra-cellular trafficking and fate of the vector genome, and has dramatically improved the gene transfer performance of AAV vectors in certain tissues.

Our current work builds on recent success in addressing the challenge posed by the species and cell type specificity of AAV capsid variants using sequence shuffling and directed evolution in the chimeric mouse human liver (Lisowski et al, Nature 2014) [6].

We are using this, and similar, approaches to select for novel AAV variants on clinically relevant cells/tissues to address number of paediatric and adult conditions (genetic and acquired), including metabolic liver disorders, urea cycle disorders, blindness, neurological disorders and disorders of the hematopoietic system.

Simultaneously we are putting a lot of effort into improving the basic AAV shuffling technology. We are running number of exciting projects addressing various aspects of AAV library function, packaging and selection processes.

For example, on the library packaging end we have designed a packaging scheme that allows us to address AAV cross-packaging issue, an undesirable phenomenon where AAV virion packages incorrect AAV genome encoding different virion, leading to the loss of connection between the genotype and the phenotype. We are also developing tools that allow us to gain a quick and unbiased insight into the pool of AAVs being selected, without the need for laborious and expensive Sanger sequencing.

We are evaluating new parental AAV variants that could be incorporated into our libraries as well as novel technologies, such as cell-specific ligands or DARPins.

Despite our interest in development of novel AAV variants, we are also constantly evaluating currently available AAV serotypes for novel applications. For example, we are interested in the correlation between AAV serotype and the immunological response (in an animal models and in humans) that can lead to clearance of therapeutic vectors, corrected cells, and prevent vector re-administration.

In a collaborative effort, TVG is involved in projects on identification and development of new clinical models that would allow to better recapitulate the human disease phenotype and obtain preclinical data more predictive of future outcomes from clinical studies. This involves comparison of various AAV variants, Physical and Transduction titres, and screens transduction of primary human hepatocytes.

Genome editing technology, most notably user-designed nucleases, are creating tremendous excitement and ushering in what many believe will be a golden age of genome engineering [7]. Their development stemmed from studies on semi-independent nuclease domains and user-targetable DNA binding proteins, giving rise sequentially to zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and engineered homing endonucleases (meganucleases) [7, 8]. The most recent development, arising from research into bacterial adaptive immunity, is the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-Cas nuclease system [9, 10].

This system represents a quantum advance, as endonuclease targeting is based on Watson-Crick base pairing between a readily designed guide RNA sequence and the target DNA sequence. The pros and cons of each technology are application-dependent with considerations including ease of design, targeting efficiency and specificity (including off-target events [1, 2], and capacity for vectorization [8].

These technologies have stand-alone utility when the desired outcome is the introduction of a disruptive mutation at a defined genomic locus, as repair of double strand breaks (DSBs) occurs through the error-prone non-homologous end joining pathway, which has been shown to cause unwanted chromosomal translocations and gross chromosomal deletions [1, 2]. From a therapeutic perspective this approach could be used to knock out a disease causing dominant allele, but more precise editing outcomes, such as repair of a mutant recessive disease locus, requires the availability of a DNA template to facilitate repair of the DSB with gene correction by the HR pathway. At low efficiency the repair template might be provided endogenously by the other allele, but higher efficiency and more sophisticated editing, such as the insertion of a selection cassette, necessitates the delivery of an exogenous template. Depending on the specific target cell type and therapeutic context, the efficiency of template delivery and gene targeting become critically important variables. Herein resides the significance and special promise of AAV-mediated gene targeting in genome editing for therapeutic purposes.

As described in “Novel AAV Variants” section above, TVG uses AAV shuffling technologies to select for AAV variants with novel properties. The selection pressure applied necessitated expression of virus encoded genes (transduction), and it has previously been assumed that the transduction performance of individual capsid variants in a specific target cell type directly predicted the performance of the same capsid variant in applications involving AAV-mediated HR. Thus variants selected for their superior transduction profile were also considered to be the best candidates for AAV-based gene editing.

In a collaborative project with a dermatology group at Stanford University, I have recently shown, definitively, that this assumption was flawed [11]. Side-by-side comparison of multiple AAV variants for their transduction and genome editing efficiencies showed that the two events were independent and transduction was not a good predictor of AAV-mediated HR efficiency.

These observations have major implications for our understanding of how capsid biology influences the intracellular trafficking and fate of the introduced vector genome. Importantly, the same data clearly showed that by performing appropriate screens we may be able to select AAV variants that would allow for higher than previously observed frequency of gene editing via HR.

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7) Segal, D.J. and J.F. Meckler, Genome engineering at the dawn of the golden age. Annu Rev Genomics Hum Genet, 2013. 14: p. 135-58.
8) Humbert, O., L. Davis, and N. Maizels, Targeted gene therapies: tools, applications, optimization. Crit Rev Biochem Mol Biol, 2012. 47(3): p. 264-81. 3338207
9) Cong, L., F.A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P.D. Hsu, X. Wu, W. Jiang, L.A. Marraffini, and F. Zhang, Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23. 3795411
10) Hsu, P.D., D.A. Scott, J.A. Weinstein, F.A. Ran, S. Konermann, V. Agarwala, Y. Li, E.J. Fine, X. Wu, O. Shalem, T.J. Cradick, L.A. Marraffini, G. Bao, and F. Zhang, DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013. 31(9): p. 827-32. 3969858
11) Lisowski, L., S.P. Melo, E. Bashkirova, H.H. Zhen, K. Chu, D.R. Keene, M.P. Marinkovich, M.A. Kay, and A.E. Oro, Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant. Mol Ther, 2014. 22(4): p. 725-33. 3982486

In a collaborative effort with CMRI Gene Therapy Unit, TVG is working on developing a number of convenient and unbiased in vitro and in vivo HR models that will allow us to gain a better understanding of the molecular mechanisms involved in vector-based gene editing by homologous recombination (HR). Those models will also serve as an ideal screening platform to evaluate HR potential of various gene editing technologies, including vector-based HR.

As the development of efficient and safe tools that will allow us to perform molecular surgeries at the genome level require an intimate understanding of the interactions between target cells/genome and gene editing tools, we are performing a screen to identify the main molecular players involved in the AAV-HR process. This knowledge will allow us to design not only more effective tools, but more importantly, safer tools that could be used in basic science and preclinical studies, but could also directly help patients by entering the clinical pipeline.

AAV Related Resources

• Primary Publications and Reviews of Interest
• Useful Online Resources

Gene Editing Related Resources

• Primary Publications and Reviews of Interest
• Useful Online Resources

AAV Production
AAV CsCl PEI Production
AAV CsCl PEI Transfection Template
AAV Iodixanol PEI Production
AAV Iodixanol PEI transfection template

AAV Titration
AAV qPCR titration protocol

Ad Production
Adenoviral vector production

Ad Titration
Ad ELISA Template file
Ad ELISA Titering
Ad ELISA VR-1516
Ad qPCR Titration protocol
Clontech Rapid Ad titration 632250

LV Production
LV CaCl2 Production
LV CaCl2 transfection template
LV PEI Production
LV PEI Transfection template

LV Titration
LV Titration by RT-qPCR
LV Titration by Transduction FACS
LV Titration by Transduction qPCR

Retro Production
Retrovirus Production
Retrovirus transfection template

We are currently recruiting for the position of postdoctoral fellow. Interested applicants should submit CV and Cover Letter to Dr. Leszek Lisowski ([email protected]).

PhD and honours positions are available. Potential applicants should contact Dr. Leszek Lisowski ([email protected]) for specific project details.

TVG is the first laboratory at CMRI to participate in newly established CMRI-UCL Bridge program that allows PhD students to enrol at either of the two institutions and perform their PhD studies in another laboratory at the partnering institution.

The CMRI-UCL Bridge Program is a great opportunity to:

• gain access to top research groups at both institutions,
  
  
• get exposed to cutting edge science in Australia and U.K.,
  
  
• build professional network,
  
  
• secure postdoctoral position at partnering institution,
  
  
• travel/have fun while learning
  
  
  

Contact: [email protected] to learn more

If you want to request a reagent, want to ask about specific unpublished results or want to initiate collaboration, please do not use our social site but send us an email at: [email protected]

You can use our Facebook page to initiate discussion with TVG team members and other researchers visiting our site, or post materials/news related to AAV gene therapy and gene editing technologies.

Other Social Sites

Facebook: @CMRI.VGEF.TVG (https://www.facebook.com/CMRI.VGEF.TVG)
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LinkedIn: https://au.linkedin.com/in/llisowski
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