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Research Overview

Our research interests are related to two main areas: 1) development of new gene therapy vectors based on Adeno-Associated Virus (AAV) as tools for tissue-specific gene delivery; and 2) development of new, safer genome-editing tools not dependant on endonuclease activity. We are developing these tools to target a broad array of tissue types and thus address a range of diseases. Although our main interests are in paediatric genetic and acquired liver disorders, we are also actively developing novel therapeutic tools for such applications as gene therapy in the CNS, eye, and haematopoietic system.

Lab Head

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Leszek Lisowski

Unit Head, Translational Vectorology Unit
Available for Student Supervision

Unit Head, Translational Vectorology Unit

View full bio

Team Members

Matthieu Drouyer
Research Officer
Kim 2
Kimberley Dilworth
PhD Student
Sophia Liao
Sophia Liao
Senior Research Assistant
Patrick Wilmott
Patrick Wilmott
Research Officer
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Deborah Nazareth
PhD Student
Maddison Knight
Maddison Knight
Research Assistant
Ramon Roca Pinilla
Ramon Roca-Pinilla
Research Officer
Carolin Von Lupin
Carolin Von Lupin
Research Officer
Suzanne Scott 1
Suzanne Scott
Research Officer
Andrea CMRI
Andrea Perez-Iturralde
Research Officer
Florencia min
Florencia Haase
Research Officer
Daniel 1 min
Daniel De los Reyes Helices
PhD Student
Steven D
Steven Devenish
Research Officer
Hardik Parate
Research Assistant
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Carmen Laura Garreta Celemin
Research Assistant
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Angela Eulalia Fornell Gutierrez
Research Assistant

Research Projects

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.

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.

More Information on Research Areas

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.

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

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.


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.

Students and Post-docs

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 (

Former Team Members

Arkadiusz Rybicki, MS, Research Assistant

Jason Ward, PhD Student, University of Sydney

Matteo Frenco, Exchange Master’s Student, Wageningen University, the Netherlands

Tessy Hicks, Exchange Master’s Student, Wageningen University, the Netherlands

Jill Muhling, PhD, Research Officer

Kate Van Brussel, Honours Student, University of Sydney

Elijah Lake, Honours Student, University of Sydney

Wojciech Kuban, PhD, Research Officer

Julius W. Kim, PhD, Research Officer

Mia Nguyen, Research Assistant 

Christina Lee, Honours Student, University of Sydney

Oselyne Ong, PhD, Research Officer

Grober Baltazar Torres, Research Assistant

Shen Goh, Honours Student, University of Sydney

Jingwei Chen, Research Officer 

Charlie Morgan, Research Officer 

Yijun Lin, Research Assistant

Inna Navarro, Research Assistant

Jessica Merjane - PhD Student

Adrian Westhaus - Research Officer

Santiago Mesa Mora - Research Assistant

Anna Koudrina, Research Officer

Jakob Kuriakose, PhD Student

Elena Barba Sarasua, Research Assistant

Marti Cabanes Creus, Senior Research Officer

Deborah Chandra, Research Assistant


RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice.

Valdmanis PN, Gu S, Chu K, Jin L, Zhang F, Munding EM, Zhang Y, Huang Y, Kutay H, Ghoshal K, Lisowski L, Kay MA. Nature Medicine 2016 May;22(5):557-62

Adeno-associated virus serotypes for gene therapeutics.

LisowskiL, Tay SS, Alexander IE. Curr Opin Pharmacol. 2015 Aug 17;24:59-67

Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.

Sebastiano V, Zhen HH, Haddad B, Bashkirova E, Melo SP, Wang P, Leung TL, Siprashvili Z, Tichy A, Li J, Ameen M, Hawkins J, Lee S, Li L, Schwertschkow A, Bauer G, LisowskiL, Kay MA, Kim SK, Lane AT, Wernig M, Oro AE. Science Translational Medicine. 2014, 6(264):264er8

Second Generation Codon Optimized Minicircle (CoMiC) for Nonviral Reprogramming of Human Adult Fibroblasts.

Diecke S, Lisowski L, Kooreman, N. G., and Wu J.C.. Methods in Molecular Biology, 2014, 1181: 1-13

Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Recapitulates Long QT Phenotype for Drug Testing.

Wang Y, Liang P, Lan F, Wu H, Lisowski L, Gu M, Hu S, Kay MA, Urnov FD, Shinnawi R, Gold JD, Gepstein L, Wu JC., Journal of the American College of Cardiology. 2014 Aug 5;64(5):451-9

Somatic Correction of Junctional Epidermolysis Bullosa by a Highly Recombinogenic AAV Variant.

Melo SP, Lisowski L, Bashkirova E, Zhen HH, Chu K, Keene DR, Marinkovich MP, Kay MA, Oro AE., Molecular Therapy, 2014, 22(4): 725-33 (*co-first authors)

Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.

Lisowski L, Dane AP, Chu K, Zhang Y, Cunningham SC, Wilson EM, Nygaard S, Grompe M, Alexander IE, Kay MA., Nature, 2014, 506(7488): 382-6

The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA.

Lisowski L, Elazar M, Chu K, Glenn JS, Kay MA., Nucleic Acid Research, 2013, 41(6): 3688-98

rAAV-mediated tumorigenesis: still unresolved after an AAV assault.

Valdmanis PN, Lisowski L, Kay MA., Molecular Therapy, 2012, 20(11): 2014-7

Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression.

Lisowski L, Lau A, Wang Z, Zhang Y, Zhang F, Grompe M, Kay MA., Molecular Therapy, 2012, 20(10): 1912-23

AAV vectors containing rDNA homology display increased chromosomal integration and transgene persistence.

Wang Z, Lisowski L, Finegold MJ, Nakai H, Kay MA, Grompe M., Molecular Therapy, 2012. 20(10): 1902-11

Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging.

Wang Y, Zhang WY, Hu S, Lan F, Lee AS, Huber B, Lisowski L, Liang P, Huang M, de Almeida PE, Won JH, Sun N, Robbins RC, Kay MA, Urnov FD, Wu JC. Circulation Research, 2012, 111(12):1494-503

Supplying therapeutic proteins from hematopoietic stem cell derived-erythroid and megakaryocytic lineage cells.

Sadelain M, Chang A, Lisowski L., Molecular Therapy, 2009; 17(12): 1994-9

Transient in vivo beta-globin production after lentiviral gene transfer to hematopoietic stem cells in the non-human primate.

Hayakawa J, Ueda T, Lisowski L, Hsieh MM, Washington K, Phang O, Metzger M, Krouse A, Donahue RE, Sadelain M, Tisdale JF. Human Gene Therapy, 2009; 20(6): 563-72

Stem cell engineering for the treatment of severe hemoglobinopathies.

Sadelain M, Boulad F, Lisowski L, Moi P, Riviere I. Current Molecular Medicine. 2008; 8(7): 690-697

Current status of globin gene therapy for the treatment of β-thalassemia.

Lisowski L, Sadelain M. British Journal of Haematology, 2008; 114(3): 335-45

A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference.

Samakoglu S, Lisowski L, Budak-Alpdogan T, Usachenko Y, Acuto S, Di Marzo R, Maggio A, Zhu P, Tisdale JF, Rivière I, Sadelain M. Nature Biotech. 2006; 24(1): 89-94

Progress toward the genetic treatment of the beta-thalassemias.

Sadelain M, Lisowski L, Samakoglu S, Rivella S, May C, Riviere I. Ann N Y Acad Sci. 2005; 1054: 78-91

Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease.

Sadelain M, Rivella S, Lisowski L, Samakoglu S, Rivière I. Best Pract Res Clin Haematol. 2004; 17(3): 517-34

Globin gene transfer: a paradigm for transgene regulation and vector safety.

Rivella S, Lisowski L, Sadelain M. Gene Therapy and Regulation 2003; 2(2): 149-175