Commercialization and transfer assistance program

PRINCIPAL RESEARCHER: PRESENTATION AND CONTACT INFORMATION

Damian Labuda, Ph.D., D.Sc.

Professor, Pediatrics Department, Montreal University

Sainte-Justine Hospital Research Center, room B-607 b

3175 Cote Sainte-Catherine

Montreal, PQ

Canada H3T 1C5

 

Telephone: [phone] ext.3586 [sec. 3282] fax: [phone] [e-mail]

 

Damian Labuda studied biology and biochemistry at Adam Mickiewicz University in Poznan, Poland, where he also obtained his Ph.D. and D.Sc.  He received additional training in Szeged, Hungary, in Saclay, France, and in Göttingen, Germany (post-doctoral fellow of Max-Planck Institute).  His early works concerned structure-function relationship in transfer RNA, origin of the genetic code, biochemistry and physico-chemistry of nucleic acids.  Since 1982, he continued his research on RNA structure and interactions, in Cedergren’s lab in the Department of Biochemistry, University of Montreal.  In 1984 he joined the Research Center of the Sainte-Justine Hospital and the Department of Pediatrics, University of Montreal, developing DNA based diagnostics program as well as research in molecular, medical, population and evolutionary genetics.  Presently, he carries studies in human population genetics on the origins and the evolutionary history of human populations, the founder effects and the genetic history of French-Canadians.  His laboratory is also involved in genetic epidemiology studies aiming genetic bases of complex diseases, the underlying genetic models, the identification of cancer susceptibility variants as well as genetic variants influencing the disease outcome

4. DESCRIPTION OF THE TECHNOLOGY

1. Background - HPV as etiological agent of cervical cancer.

HPVs are genetically diverse; those infecting genital epithelium represent types with low and high oncogenic potential. Low-risk HPVs, such as types 6, 11, 34, 40 44 and others, cause benign genital warts, whereas high risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) lead to cervical cancer. The most frequent 16 and 18 account for about 70% of infections seen in cervical carcinoma (Bosch et al., 2002; Franco et al., 2001); HPV 16 alone being found in nearly 50% of high grade lesions. There exists a substantial degree of DNA sequence identity among types as well as between malignant and benign HPV strains. The difference between particular HPV types in their sequence segments that are relevant to the HPV molecular testing can be as small as few nucleotides (Villa et al., 2000; zur Hausen, 2000).

Cervical cancer is presently the second most common cancer affecting women worldwide, and the most frequent malignancy among women in developing countries (Ferenczy & Franco, 2002). It was estimated that 493,000 new cases of invasive cervical cancer were diagnosed worldwide in 2002, representing nearly 10 % of all cancers in women (Ferenczy & Franco, 2002). Indeed, the HPV-attributable proportion of the cervical cancer is estimated at more than 95%. The relative risk associating HPV infection to cervical neoplasia is very high and increases to several hundred in the case of the persistent infection with types 16 and 18 (Bosch et al., 2002; Clavel et al., 2001; Franco et al., 2001). Once molecular pathogenesis of cervical cancer was recognized, it became clear that an accurate type-specific testing of HPV is absolutely required for the disease prevention and management, as recurrent detection of high-risk HPV types is a strong predictor of high grade cervical intraepithelial neoplasia (CIN) and invasive cancer lesions. (Brummer et al., 2006; Schlecht et al., 2001; Trottier & Franco, 2005) Importantly, in the era of HPV vaccination, planning cervical cancer screening will inevitably have to change, emphasizing need for HPV type-specific assays. (Franco et al., 2006). Several diagnostic kits are commercially available and numerous diagnostic systems have been described. However, of significant importance is the recent study by the World Health Organization (WHO) which carried detection of 24 samples of seven most frequent HPV types, using commercial individual typing kits, such as PGMY line blot (Roche), SPF10-LiPa (Innogenetics), Deg GP5+/6+ reverse line blot and DNA chip (Biomed Lab Seoul, Korea). The measurements were performed in 29 independent laboratories and 12 different countries. The overall detection rate of HPV16 was 62% and that of HPV18 was 73.9%; approximately, half of the laboratories failed to identify HPV type 6. In 2008, WHO issued recommendation guidelines for the use of “reconstructed” clinical samples and therefore make a common cross-reference among different diagnostics tests and different platforms on the identically “reconstituted” samples.

2. Technology developed and scientific basis

Four years ago we proposed a novel and generally applicable approach consisting of the development of nucleic acid probes by selection in vitro. This differs from commonly used approaches based on the rational design of probes. Addressing the common problem of DNA diagnostics to distinguish between closely related population variants, similar strains or subtypes, we developed a novel technology that would allow generation of probes discriminating DNA targets that differ only by few sequence positions. Specific probes are selected from a random oligonucleotides mixture by a process of iterative hybridization. Repetitive rounds of forward and subtractive hybridization lead to specific pools of probes with high discriminatory power from which individual probes can be cloned.

In the summer 2004, we obtained the funding from CIHR to develop this technology and show its applicability using a model system of HPV infection. The first task was to develop proposed technology. For that, we used a specific segment of the viral L1 gene of six HPV types differing by one to seven sequence positions. Starting from the initial pool of probes we obtained pooled probes, and subsequently, target-specific cloned probes. Both pooled and cloned probes were shown to discriminate well between six HPV types. (Brukner et al, NAR, Brukner et al, Nature protocols, 2007). Once the technology was developed, we extended the experiments to 39 HPV types. For the selection we used the HPV genome region corresponding to the PCR amplified HPV segment usually used in the clinical diagnostics (GP5+/6+ region of the viral L1 gene). We have obtained a set of 39 probes specific for each of the targeted HPV type and efficiently discriminating against the remaining 38 types in a single experiment under the ambient temperature of hybridization (Brukner et al., J. Clinical Virology, 2007).
In summer 2007 we obtained proof of principal grant from CIHR to simplify our assay and render it generally applicable in the research and diagnostic setting. We tested the probes in the reverse format (as opposed to previously used direct format) in which the array of the HPV specific probes is immobilized on solid support. This format is more suitable for diagnostic use and allows simultaneous detection of distinct HPV variants in the clinical sample. Miniaturization of the assay was achieved by single compartment hybridization (as opposed to previously used 96-well format). For this, the attachment of probes was performed via streptavidin-biotin connection using SAM membranes (Promega, WI). For all testing we used full-length GP5+/6+ target oligonucleotides (and double strand amplicons derived from these oligonucleotides, mimicking thus PCR from clinical samples) labeled with [³²P] for initial optimization (Fig 1).

HPV 6

HPV 11

HPV 16

HPV 18


Figure 1. Example of HPV typing probes in reverse format. Hybridization signal between 39 selected HPV type-specific probes (each 100 pmols), y spotted on SAM membrane (7 cm x 3 cm) and particular intended targets (HPV 6, HPV 11, HPV 16 and HPV 18 on upper panel and HPV 31, HPV 33, HPV 35, HPV 39 HPV 51 and HPV 68, on lower panel) each presented in the concentration of 3 pmols per 5 mL of hybridization solution. (P32 autoradiography). Hybridizaton is performed at ambient temperatue (26+/-4^oC) using buffers compatable with colorimetric detection. No false positives (cross-hybriization) with remaining 38 HPV types was seen.

As a result of these successful developments, we subsequently focused our assay on four most relevant HPV types (6, 11, 16 and 18). These types are the most common worldwide and the most relevant from the point of view of recently introduced national vaccination programs (duration and liability of vaccination strategy can be monitored). At the same time, we can guarantee that probes that we obtained during selection will not produce significant cross-talk with other relevant HPV types as shown in Fig. 2.

Figure 2 – Hybridization signal (y-axis) between selected 6-FAM labeled type-specific probes (HPV 6, 11, 16 and 18, using 20 pmol of each) and 39 HPV GP56 targets, where HPV types-specific numbering nomenclature is ranked in the growing order on x-axis. Each target (20 pmol) is bound to one well (using 96-well steptavidin-coated Pierce plate) and hybridization is performed in 100L volume (see Brukner et al, 2007, J. Clinical Virology, for more details).

3. Current development status of the technology – functional prototype

Processes/algorithm for the generation of spectrum of specific nucleic acid (NA) probes able to discriminate against plurality of similar targets is not yet known. When sequences that are to be distinguished are similar, the difference in their binding energy is small, restricting the window of adjustable experimental conditions, which would allow discrimination between all potentially reacting species. Finding such conditions is usually problematic in multiplex applications, when many probes and/or many targets are considered simultaneously. Moreover, if probes with such a requirement have to perform under robust conditions, the design process is especially prone to failure. Here the term “specificity” is used as an ability to discriminate NA target in the context of similar NA targets (differentiating up to 87% sequence identity), while assay is considered “robust” if stability of the assay performance within a wide range of performing conditions is preserved.

Following urgent need for developing more robust and more accurate HPV typing assay, we applied this approach to obtain new generation of probes. These probes are selected to discriminate among 39 clinically relevant HPV types, based upon the previously characterized GP5+/6+ L1 segment of the HPV. In a series of hybridization steps, starting from a mixture of random oligonucleotides, we iteratively enriched mixture in oligonucleotides that selectively recognize each specific HPV type out of the 39 HPV targets. (Brukner et al, NAR, J Clin Virol, Nature protocols, 2007). A detailed analysis of data showed clearly that, given the number of variables, the rational design of probes would not be as efficient and straightforward as selection performed in vitro. Analysis of sequences of obtained probes showed that specificity of binding between probes and targets is achieved through a fine balance between non consecutive stretches of base-pairs, segments of mismatches and often accommodation of secondary structures. These combinations allow maximizing the difference in binding energy between probe-specific target and probe-unspecific target complexes.
In the next phase, we optimized a reverse format of probes (Fig 3). The performance of the assay was examined using clinical samples containing HPV 16 and HPV 6.




Figure 3. HPV typing of pre-characterized clinical samples containing HPV6 and HPV16 to the array of 39 immobilized type specific clonded probes (CP). (A) the arrangements of CP probes; (B) hybridization with HPV6; (C) hybridization with HPV16. Arrows indicate the orientation of the probes array. (see Brukner et al, J Clinical Virol. 2007, for more details)

We confirmed specificity of our probes and stability of our novel assay at the wide range of temperatures, starting from 20°C to 28°C and in the different spectrum of non-denaturing buffers (classical hybridization buffers, as well as simple, PCR-like and colorimetric-compatible buffers).

Such assay performance is prerequisite for the future point-of-care medical device, contrary to the present genotyping assays, whose setting performance is not only challenging for similar NA targets, but also based on sophisticated technological platforms.

4. Scientific value

The main advantage of our method resides in its enhanced power of identification and discrimination between multiple short nucleic acid sequences that differ by a few mutations, as it is the case with different HPV types, in a multiplex hybridization assay. In other words, instead of adjusting hybridization conditions to the whole set of probe: target pairs that we want to include in the diagnostic device, by using iterative hybridization we adjust the probes to the conditions we have chosen.

Example of direct expected outcome using our HPV typing assay in clinical follow-up is to estimate HPV vaccine performance and its protective time in industrial countries where HPV vaccine program is available. It will also provide a cost-effective but accurate and easy to use diagnostic tool, an essential requirement for deployment of HPV screening program in developing countries.
Finally, the methodology of probe selection is applicable to many other medical conditions where investigation or diagnosis or detection of resistant strains is based upon a differentiation of highly similar nucleic acid sequences (HIV, hepatitis virus, tuberulosis..).

5.