Finding cures for children's genetic diseases

For Researchers


Modern genomics offers unprecedented prospects for both discovery science and human health. Major health impacts will be achieved through gene and cell therapy approaches, individually and in combination, but the technologies required are only just beginning to come of age. The three critical challenges for the gene therapy field are:
  • improving the efficiency with which cell populations can be targeted,
  • developing tools that allow precise gene modifications without affecting physiological gene expression and control,
  • avoidance of inadvertent damage to the genome with the associated risk of neoplasia.
Fully addressing these challenges requires the development of precise, specific and highly efficient cell targeting and genome editing technologies.
Due to their non-pathogenic character, ease to generate and mostly episomal character, vectors based on Adeno-Associated Virus (AAV) have become the vectors of choice for many gene therapy applications. Their preferential liver-tropism and low preference for other tissues and cell types remains a significant obstacle we still need to overcome.
On the genome engineering side, much justified fanfare has accompanied the development of user-designed nucleases, but the therapeutic potential of this evolving technology remains constrained by the challenges associated with delivery, immunogenicity, and significant off-target activity [1, 2], and they are highly disease- and target cell type-specific [3].
The scientific interests of Translational Vectorology Group (TVG) focus on understanding the basic biology behind AAV-mediated gene editing and further developing the synergistic therapeutic potential of AAV-based gene addition and AAV-mediated gene targeting technology, which simultaneously addresses the challenges of efficient delivery and precise genome editing.
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.

Novel AAV variants
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.

AAV Vectors
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.

Animal Models
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.
HR-screen models
In 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.
AAV vs Genome (Efficiency and Safety)
As development of efficient and safe tools that will allow us to perform molecular surgeries at the genome level require 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 clinical pipeline.
1.         Cho, S.W., S. Kim, Y. Kim, J. Kweon, H.S. Kim, S. Bae, and J.S. Kim, Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res, 2014. 24(1): p. 132-41. 3875854
2.         Cradick, T.J., E.J. Fine, C.J. Antico, and G. Bao, CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res, 2013. 41(20): p. 9584-92. 3814385
3.         Lisowski, L., S.S. Tay, and I.E. Alexander, Adeno-associated virus serotypes for gene therapeutics. Curr Opin Pharmacol, 2015. 24: p. 59-67.
4.         Logan, G.J. and I.E. Alexander, Adeno-associated virus vectors: immunobiology and potential use for immune modulation. Curr Gene Ther, 2012. 12(4): p. 333-43.
5.         Wu, Z., A. Asokan, and R.J. Samulski, Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther, 2006. 14(3): p. 316-27.
6.         Lisowski, L., A.P. Dane, K. Chu, Y. Zhang, S.C. Cunningham, E.M. Wilson, S. Nygaard, M. Grompe, I.E. Alexander, and M.A. Kay, Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature, 2014. 506(7488): p. 382-6. 3939040
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