Upcoming conferences

Dr Alison Todd will be attending the following conferences in Feb 2012:

Quantitative Real-Time PCR for Molecular Diagnostics

16-17 February 2012

San Francisco, CA, USA

Molecular Medicine Tri Conference

19-23 February 2012

San Francisco, CA, USA

Conferences attended

Next Generation Molecular Diagnostics

1-3 August 2011

San Francisco, CA, USA

Bio International Convention

27-30 June 2011

Washington DC, USA

AusMedtech 2011
23-24 May 2011
Sydney, NSW, Australia

Molecular Medicine Tri-Conference
23-25 February 2011
San Francisco, CA, USA

Use of MNAzyme qPCR in an anti-HIV gene therapy clinical trial
Gozar, Mary Margaret(2), Todd, Alison V(1), Symonds, Geoff(3), Fuery, Caroline J(1)

13-15 February 2011
(1) SpeeDx Pty Ltd, National Innovation Centre, 4 Cornwallis St, Eveleigh NSW 2015 Australia. (2) St George Hospital, Clinical Trials Unit, Ground Floor, WR Pitney Bldg, St George Hospital, Gray Street, Kogarah, NSW 2217. (3) St Vincent’s Centre for Applied Medical Research: Level 8, Lowy Packer Building, 405 Liverpool St, Darlinghurst, NSW 2010, Australia.

Background

MNAzymes are multicomponent nucleic acids that can be linked with PCR to provide highly specific and robust real time detection of amplicons. Two “partzymes”, composed entirely of DNA, assemble on a PCR amplicon to produce a catalytically active MNAzyme that cleaves a universal reporter probe (Mokany et al. 2010 JACS 132:1051-1059).

MNAzyme qPCR was used to follow the levels of expression of an anti-HIV cell-delivered gene therapeutic (OZ1) in a multi-centre international Phase II clinical trial. OZ1 is a retroviral construct based on the murine retroviral vector LNL6 containing an anti-HIV ribozyme. Seventy four subjects were infused with OZ1- or placebo-transduced autologous CD34+ hematopoietic progenitor cells and then followed for two years. Details of the placebo-controlled double blinded trial have been published elsewhere (Mitsuyasu et al. 2009 Nature Medicine 15, 285-292).

Methods

The MNAzyme qPCR assay for OZ1 expression was validated and performed in a Central Laboratory setting. Measurement of OZ1 was duplexed with measurement of a human endogenous control. Multiple controls were included with each performance of the assay and included positive controls with three different amounts of OZ1, negative controls with no OZ1 RNA, and negative controls with no RNA.

Clinical samples were received and tested over a 2.5 year period. Samples assessed for OZ1 expression included peripheral blood mononuclear cells (PBMC) and isolated samples of granulocytes, CD4+ T cells, CD8+ T cells, B cells, monocytes, CD4+/CD45+ T cells, CD4+/CD45- T cells, CD8+/CD45+ T cells, and CD8+/CD45- T cells. Levels of OZ1 expression were only measured in patients in the treatment arm.

Results

OZ1 expression appeared to decrease over the two years of assessment post infusion. The percentage of patients showing detectable levels of OZ1 expression dropped from 100% immediately post-infusion to approximately 30% by week 100. Overall approximately 0.6% of patient samples produced quantitative results, 38% showed detectable results that were not quantitative, and 61% of samples had levels of OZ1 RNA that was below the limit of detection of the assay.

There was no signal from any negative control in any of the 84 performances of the assay. Results from the positive controls showed stable and robust measurement over the 2.5 year testing period.

Conclusions

Lorne Genome Conference
13-15 February 2011
Melbourne, VIC, Australia

AusBiotech 2010 National Conference
19-22 October 2010
Melbourne, VIC, Australia

7th Australasian Viral Hepatitis Conference
6-8 September 2010
Melbourne, VIC, Australia

MNAZYME qPCR USED TO QUANTIFY MULTIPLE GENOTYPES IN HEPATITIS C
INFECTION.
Jacka BJ^, Mokany E#, Pham ST*, Carrera A**, Kelleher AD***, Matthews GV^, Dore GJ^,
Applegate TL^
^ National Centre in HIV Epidemiology and Clinical Research, University of New South
Wales, Sydney, NSW, Australia. # SpeeDx Pty Ltd, Eveleigh, New South Wales,
Australia 2015. * School of Biotechnology and Biomolecular Sciences, University of
New South Wales, Sydney, NSW, Australia. ** NSW State Reference Laboratory HIV /
AIDS, St Vincent’s Hospital, Sydney, NSW, Australia *** Immunolgoy Laboratory, St
Vincent’s Centre for Applied Medical Research, Darlinghurst, NSW 2010, Australia.

Research into optimization of current and future therapeutic strategies for HCV is presently hindered by limitations in the sensitivity and accuracy of available assays. In particular, the prevalence and significance of mixed HCV infection in high risk populations are poorly understood. Assays that are better able to characterise the true prevalence, dynamics and impact of mixed HCV infection will enhance research into viral clearance, cross-protection and vaccine development. This project aims to develop a method to quantify and detect mixed HCV infection using MNAzyme quantitative PCR (qPCR). MNAzymes are multi-component nucleic acid enzymes composed of two DNA “partzymes” which come together to create catalytically active non-protein enzymes, in the presence of specific nucleic acid target. The catalytically active MNAzyme then binds and cleaves a universal probe. MNAzymes and fluorescently labelled universal probes can be adapted to quantify any nucleic acid target during qPCR. The well characterized probes facilitate the ability to multiplex and quantify several targets simultaneously. Consensus primers targeting the Core region were designed to amplify four HCV genotypes (1a, 1b, 2a & 3a). Partzymes were designed to specifically target each of the four genotypes, each designed to recognize and cleave a different fluorescently labelled universal probe. Viral RNA from HCV+ patient serum was extracted, transcribed and amplified by nested RT-PCR. MNAzyme qPCR demonstrated high specificity and effectively discriminated between each genotype. MNAzyme qPCR demonstrated a linear range over a minimum of 4 orders of magnitude (r2=0.9988). The MNAzyme qPCR method will be further
developed and validated and used to determine the true prevalence and kinetics of mixed HCV in early infection.

Cambridge Healthtech Institute

Xgen Congress

Sixth Annual Quantitative PCR Conference

March 17-19th 2010
San Diego, CA, USA

MNAzyme qPCR Technology has Superior Multiplexing Capabilities and Provides a Novel Detection Strategy for SNPs
Alison Todd, Ph.D., Chief Scientific Officer, SpeeDx Pty Ltd. Thursday March 18 2010
We have developed a new qPCR technology that utilizes novel non-protein enzymes known as "MNAzymes". The catalytically active MNAzymes cleave generic reporter probes and generate fluorescence. The use of generic probes, as opposed to target specific probes, provides multiple advantages over other qPCR methods. A novel method for single base discrimination has been further developed and utilized for SNPs. For all applications, MNAzymes have a superior capacity for multiplex analysis, due to their robust nature and cost effectiveness.

Mokany E., Doan T.B., Young P.E., Suwandi R., Lomas M., Wang L. and Todd A.V. A new real-time PCR technology mediated by Multi-component Nucleic Acid enzymes (MNAzymes) has advantages over other real time chemistries. Quantitative PCR: replicating and validating success. April 2008. San Diego

Mokany E., Doan T.B., Young P.E., Suwandi R., Lomas M., Wang L. and Todd A.V. A novel detection technology mediated by multi-component nucleic acid enzymes (MNAzymes) allows rapid development of multiplex real-time PCR assays using generic probes. 40th Annual Oak Ridge Conference Breakthrough Technologies for Clinical Diagnostics April, 2008 San Jose, CA, USA

Mokany E. Multi-component Nucleic Acid enzymes, a new real-time PCR technology. 5th Annual Australian QPCR user meeting. Garvan Institute, Sydney, Australia and Melbourne University, Melbourne, Australia, April 2008

Mokany E., Doan T.B., Young P.E., Suwandi R., Lomas M., Wang L. and Todd A.V. A new real-time PCR technology, which is mediated by Multi-component Nucleic Acid enzymes (MNAzymes), has a superior capacity for multiplex analysis. CHI Quantitative PCR, Microarrays and biological validation Providence, Rhode Island, USA, October 2007