Commercialization and transfer assistance program
Yüklə 0,62 Mb.
|
- Bu səhifədəki naviqasiya:
- Previous disclosure and/or anterior art
- Method of selection probes
- Research report on the PCT patent application
- Freedom to operate analysis (FTO)
- Acquired or planned protection
- TECHNOLOGY DEVELOPMENT PLAN
- Identification of current stage of technology development.
- Remaining Steps and Time to Market
- Technical and technological challenges to be met and anticipated progress
- Type of activities to be carried out
- Clinical validation of the prototype assay
- Design of the commercial format of the assay
- Establish the regulatory pathway for the commercial use of the assay
- Optimization of the commercial format of the assay for high sensitivity and specificity
- Validation of the performance of the commercial assay with clinical samples.
- Work plan and timetable
- Deliverables and result indicators
- Decision-making milestones (go/no go) for measurable and clearly identified results
- Proposal for granting the subsidy according to the decision-making milestones reached
- ESTABLISHMENT’S PROJECT MANAGER (In French)
INTELLECTUAL PROPERTY
In January 2006, Dr. Brukner, Dr. Labuda and Dr. Krajinovic filed an invention disclosure at the research administration of Hôpital Sainte-Justine. The inventorship’s contribution is presented in Table 1.
Table 1. List of the inventors and their contribution to the invention The invention describes a new method for generating sequences of hybridization probes, suitable for multiplex target detection, even if the plurality of targets are very similar at the sequence level. The method is based on the hybridization of nucleic acids. A prototype for genotyping HPV virus was built and performance of 6 selected probes was tested and compared with complementary probes. A set of 39 oligonucleotide probes for HPV typing was generated and more recently a prototype assay was optimised for 4 probes (see section 6.1.). A provisional patent application entitled “NUCLEIC ACID PROBES, METHODS FOR THEIR PREPARATION AND USES THEREOF” was filed in the US in August 2006. We filed a complete PCT application in August 2007 and we expect to enter the National Phase in different markets in February 2009 (see section 5.3.1.). M. Serge Shahinian from the firm Goudreau Gage Dubuc in Montreal is the patent agent handling the file. The data base “Derwent World Patents Index”, where specialist editors provide a comprehensive summary of the patent contents with its advantages gives the following description of the invention: NOVELTY - Identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid comprises: (a) hybridizing the first nucleic acid with oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences; (b) amplifying the bound oligonucleotides to obtain amplified oligonucleotide duplexes; and (c) repeating hybridization in the presence of a second nucleic acid, where an oligonucleotide comprising the random nucleotide sequence can be used for discriminating the first nucleic acid from the second nucleic acid. DESCRIPTION - Identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid comprises: 1.hybridizing the first nucleic acid with a pool of oligonucleotides in a hybridization mixture, the oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences; 2.removing oligonucleotides which are not bound to the first nucleic acid from the hybridization mixture; 3.dissociating bound oligonucleotides from the first nucleic acid; 4.amplifying the bound oligonucleotides using primers capable of binding to the primer recognition sequences to obtain amplified oligonucleotide duplexes comprising a first strand corresponding to the bound oligonuc leotides and a second strand corresponding to the complement of the bou nd oligonucleotides; 5.treating the duplexes to remove or degrade the second strand to obtain single-stranded amplified oligonucleotides; 6.repeating (a) to (e), where the pool of oligonucleotides of (a) is the amplified oligonucleotides obtained in (e) thus to obtain further ampli fied oligonucleotides; and 7.repeating (a) to (e), where the hybridization in (a) is performed in th e further presence of the second nucleic acid; where an oligonucleotide comprising the random nucleotide sequence of the further amplified oli gonucleotides can be used for discriminating the first nucleic acid fro m the second nucleic acid. INDEPENDENT CLAIMS are: 1.an oligonucleotide (a) identified by the method above, (b) capable of discriminating a first nucleic acid from a second nucleic acid, where the oligonucleotide is not exactly complementary to the first nucleic acid, and (c) comprising a nucleotide sequence selected from SEQ ID NO. 1- 43, 100-104, and 116; 2.a method for detecting the presence or absence of a first nucleic acid in a sample; 3.a kit for detecting the presence of a first nucleic acid in a sample, the kit comprising the oligonucleotide; 4.a collection of two or more oligonucleotides, where the oligonucleotides comprise a nucleotide sequence selected from SEQ ID NO. 1-43, 100-104 , and 116; 5.an array comprising the oligonucleotide or the collection of two or more oligonucleotides; and 6.a kit for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, the kit comprising the pool of oligonucleotides. USE - The methods are useful for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, and for detecting the presence or absence of a first nucleic acid in a sample. The kit is for detecting the presence of the pathogen in the sample, for detection of the pathogen in the subject, and for diagnosing a disease or condition associated with the pathogen in the subject (all claimed). The methods can be used for discriminating between closely related or similar nucleic acids, and for identifying or preparing an oligonucleotide for discriminating a desired or intended target nucleic acid from other undesired or non-intended non-target nucleic ac ids. The oligonucleotides, methods, and kits may be used in analytical, diagnostic (e.g. infection of an animal, plant, or organism by a pathogen), detection, manufacturing/quality control, research, environmental monitoring (e.g. pollution/contamination of air/water/reagents) intended for use in biological systems (e.g. culture or animal systems/other materials), microbiology (detection studies of organisms difficult to c ultivate), and forensic applications. ADVANTAGE - The method has the capacity of identifying or preparing an oligonucleotide for discriminating nucleic acids, which share sequence similarities, e.g. similar nucleic acid sequences from different organisms (e.g. orthologous genes), variants (e.g. polymorphisms, different alleles) of a given nucleic acid sequence, nucleic acid sequences derived from genes belonging to the same family or nucleic acids derived from subtypes of a given organism (e. g. virus, bacteria, parasites).
In conjunction with Univalor, the organization that provides commercialization services to the research centre of Hôpital Sainte-Justine (see section 9), we performed a review of the prior art. The search was carried out using Delphion patent databases, and literature (PubMed) together with a web-based search.
The concept of selecting nucleic acid sequences that specifically bind particular targets has been developed using an approach called SELEX (systematic evolution of ligands by exponential amplification. However that patented method (see Gold, Table 2a) and other related methods described in the literature (c.f. Kyung 2003) do not teach the use of iterative selections for generation of nucleic acid ligands against nucleic acid targets for the purpose of genotyping or identifying/detecting nucleic acids. Therefore, our iterative hybridization method to select probes that can discriminate between closely related or similar nucleic acids is entirely novel.
Table 2a. Patent related to the method
Our search revealed, that prior art taught the use of nucleic acid probes to detect HPV types. Several patents (see examples in table 2b and in Annex 1) and articles (Hwang et al., 2003; Klaassen et al., 2004; Kleter et al., 1999; Schmitt et al., 2006; van den Brule et al., 2002) describe typing systems based on PCR/classical hybridization but in all cases they rely on complementary sequences to discover HPV types (hybridization rules based on Watson-Crick base-pairing). Our probes do not rely on complementary sequences and also none of our probes sequences are disclosed or claimed in these documents.
Table 2b. Patent related to our HPV assay
In November 2007, we received the international research report and the written opinion of the international searching authority. Seven documents, including four documents published by our inventors were found to oppose our invention in terms of both novelty and inventive step (nothing was opposed to the industrial applicability of the invention). We carefully analyzed these documents and our conclusion, which was validated by our patent agent, is that none of these documents teach, disclose or can be considered as anterior art of what we claim in our invention (see Annex 2 for a summary of our analysis). In conclusion, based on our analysis of the prior art, we do not anticipate any problems for the patentability of our invention and we are confident that we would be in an excellent position to argue potential objections from patent Examiners at the regional and national phases of protection, should there be any. This confidence was validated by the patent agent handling the file. It might be important to mention that Univalor’s team has experience in patent prosecution and with interpretation of PCT written search and opinion reports.
We do not see any FTO problems related to the commercial use of our new method for generating sequences of hybridization probes concerning the SELEX patent (see section 5.2.1) The SELEX process is described in two issued US Patents, No. 5,270,163 (filed Aug 1992), which claims the SELEX method itself, and No. 5,475,096 (see table 2a) which claims the use of the method to identify nucleic acid ligand for target molecules “other than a polynucleotide that binds to said nucleic acid ligand,” and “wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.” Our preliminary FTO search and analysis gives us no indication that the commercial production, marketing and use of our new assay for HPV detection would infringe the intellectual property rights of other patents. Our analysis included the US patents listed in table 2c and in Annex 1. It appears that these inventions relate mainly to the HPV DNA isolated from the appropriate strain, the probes containing these DNA sequences, and the kits containing these probes. Because the intrinsic nature of our probes is not fully complementary to any of the HPV DNA, our probes are not covered by the claims of these patents. Our position was validated by our patent agent. Note that most of the HPV types were covered by patents that are now expired or will expire in the near future. Consequently, we do not anticipate a need to license any patent on either SELEX or any specific sequence of HPV variants in order to commercialize our technology.
Table 2c US patents related to HPV types
A provisional patent application entitled “NUCLEIC ACID PROBES, METHODS FOR THEIR PREPARATION AND USES THEREOF” was filed in the US in August 2006. That patent application claims a method for identifying and preparing probes for selective detection of nucleic acids. This method is particularly useful in discriminating between closely related nucleic acid sequences. Such a method may be used in a variety of analytical and diagnostic research and related applications. Probes selected for 39 different HPV types are covered in the patent application. A complete PCT application was filed in August 2007.
We plan on entering the Regional and National phases in February or March 2009 as described in the following table (Table 3).
We will budget another patent filing in a territory that will be chosen between the following: Europe, Angola (a relevant territory for our financial partner in this application) and Brazil (the translation of the application into Portuguese for the Angola application can be subsequently used for the Brazil application and will reduce the cost of filing in this country by 50%). The IP protection in Canada, South Africa and ARIPO is justified by the partnership with the company Continental Diagnostic, based in South Africa (see section 14), and the interest of Warnex, based in Laval, Québec to provide HPV diagnostic services through Canada. It is also strategically important to get protection in US which is the largest market for HPV testing presently. Europe and Brazil are also two relevant territories for IP protection since vaccines clinical trials were done in these territories. However the final choice between Europe, Brazil and/or Angola will be decided in January 2009.
The documents attesting to the assignments of titles, rights and revenue sharing between the researchers, the CHU Sainte-Justine Research Centre (“CHU Ste-Justine”), its commercialisation entity (i.e. Valorisation-HSJ, limited partnership) and/or the Université de Montréal (“UdeM”), shall be concluded prior to the start of the project, the whole according to the provisions of the intellectual property policies and related agreements in force between these institutions. As a precision, CHU Ste-Justine is an affiliated institution of UdeM and as such, CHU Ste-Justine and UdeM jointly own, in equal parts (50%/50%), the undivided ownership rights in any invention originally disclosed at CHU Ste-Justine by a researcher who holds an academic qualification or a faculty title of UdeM. CHU Ste-Justine and UdeM also jointly own, in equal shares (50%/50%), the institutional share of the benefits or revenues which will be allotted to CHU Ste-Justine, as mutually agreed between these institutions. The share of the proceeds of commercialization of the invention (the “Proceeds”) shall therefore be divided and paid as follows:
Table 3. Final sharing of the Proceeds between the parties
The proposed development plan will allow optimizing and validating the efficacy of a new HPV assay and will provide us with convincing arguments that it can fulfill all principal requirements of clinical and market needs: multiplex detection, detection of different HPV types in the same patient (super infection), low production cost and ease of use.
The actual prototype kit is composed of 4 probes designed to detect 4 HPV vaccine- relevant types (6, 11, 16 and 18), including two positive controls: one reflecting presence of any HPV in the sample and the other controlling for sample DNA integrity (see Figure 4). HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived through amplification or chemical synthesis. 
Figure 4: Schematic presentation of 4 cm x 2cm Steptavidin-coated Membrane (SAM, Promega) with biotinilated oligonucletide probes Optimal number of picomoles (right panel) spotted on the membrane (left panel) for each HPV probe and controls (UP, universal HPV probe, CD, control DNA designed for the control of the quality of DNA extraction HPV 6, HPV 11, HPV 16 and HPV 18 are the most frequent virus types. In fact, HPV types 16 and 18 account for 70% of HPV infections. Accurate and affordable HPV genotyping for these strains will also be in high demand for the next few decades due to HPV vaccination studies. Recently, two experimental vaccines to prevent infection with HPV 6, 11, 16 and 18 became available. HPV screening is needed here: i) to identify individuals that are eligible for vaccination and ii) to monitor the efficiency of vaccines. The Advisory Committee of Immunization Practices (ACIP, US) recommended the continuation of HPV screening protocols until (i) other type-dependent vaccines are developed and (ii) the protective time period of the vaccine is fully characterized. The advantage of our assay is in the selection of probes in the context of 39 viruses. Therefore, the probes are highly specific for these types and do not cross-hybridize with other HPV amplicons (see Figure 2). We also developed an HPV universal probe, which is designed to detect any HPV infection (Fig. 5, probe UP). This “promiscuous probe” allows for the follow-up of HPV positive cases (but HPV 16 and 18 negative ones), which is of particular interest to researchers and to public health. Despite typing only 4 viruses, all cases of HPV-positive cases could be clinically registered. Further type-specific analysis can be then performed by complementary methods. Recently, we also introduced a strategy that will allow monitoring of DNA quality and quantity of a clinical sample. This strategy includes new PCR and a corresponding new probe. The probe was designed to detect monomer repeat of 29 adenines (A29), present in a single copy locus of human genome of well-described BAT26 amplicon (Fig. 5 probe CD). 
The performance of each type-specific probe and its corresponding intended targets is presented in Figure 6A. 
Class II, III and IV medical devices need to have Canadian licenses in order to be sold in Canada. After speaking with Sarah Chandler, Acting Head of the Regulatory and Scientific Section at the Device Licensing Services Division of the Medical Devices Bureau at Health Canada, we believe our HPV test is most likely a Class III product. We plan to validate the classification status of our device before the end of 2008. This is the first step toward getting a medical device license. The regulatory process in South Africa starts with a letter to the authority mentioning the intention to file clinical information for registration of a new test. The authority can call for local clinical information and trials if deemed necessary. When the information has been submitted, questions could follow on the proof of concept and clinical validation methodology results. The whole process takes up to 3 years right now, but when the new authority steps in it might be shortened to 18 months and if fast tracked, 9 months. Our strategy is to partner with a company already established in the molecular diagnostic market who will be responsible for the final stages of our product development, including all aspects of clinical validation and regulatory affairs. We will have the opportunity to work with the Quebec company Warnex (see letter of support) to define the regulatory path for our HPV assay.
1) Preservation of assay specificity and sensitivity under different assay conditions Our results indicate that we will be able to develop a new format of HPV typing kit that will preserve clinical sensitivity (genome equivalent, GE, 1000 or more), but will have unique specificity features preserved in a wider-than-usual range of assay conditions. What remains is to prove that our type-specific probes will produce better assay stability than any other probes on the market. It is well known from literature data that small variations in HPV genotyping assay conditions can lead to the wrong interpretation of hybridization intensity patterns when using known commercial kits. The most critical issues are in the domain of specificity of hybridization among multiplicity of similar targets, where even a 1oC deviation from the intended temperature, or small variations in buffer conditions can be detrimental. In fact, recent data (Journal of Clinical Virology 42 (2008) 412–414) showed that even intra-laboratory repetitions with the same clinical samples (but different DNAextractions) did not produce the same results using Roche Linear Array assay (83% concordance). We believe that our HPV typing probes have inherent (sequence-dependent) features which preserve stability under a wider range of assay conditions. Considering that this issue presents a major barrier to the current accuracy of hybridization-based assays, overcoming it by confirming our data in a clinical setting would be important accomplishment. As an example of anticipated progress, we recently perform hybridizations between HPV 6, 11, 16 and 18 single stranded targets (GP5+ strand) at 4 different temperatures (20, 25, 30 and 35oC) and recorded hybridization pattern, as presented on Figure 6B 
We already have a functional prototype assay format which will be challenged with reconstructed samples having 1,000 and 10,000 genome equivalents (GE) of different types of HPV in the background of 1,000 GE of human DNA (see Figure 7). 
We do not foresee obstacles in detecting 1,000 genome equivalents of HPV in the hybridization assay. In fact, our present developments indicate that a hybridization assay is not an obstacle per se, since 1,000 GE of HPV 6, 11, 16 and 18 are amplifiable by PCR (using oligonucleotide targets as substrates) in sufficient quantity to guarantee detection of the hybridization signal. We will have to do HPV GP56 amplification in the context of human DNA, where we will have to satisfy “normal” PCR yield requirements (1-10pmols) and at the same time be able to perform CD PCR (i.e. produce amplicons which reflect DNA quality of sample). 
3) PCR amplification from clinical samples Protocols designed to collect, store and purify DNA from cervical swabs are well-described. And the expertise of Dr Gòrska-Flipot Izabella from the Hotel-Dieu diagnostics laboratory, guarantees that this challenge will be addressed in a professional, effective fashion given the long clinical experience and expertise she has in the domain of molecular diagnostics. 4) Technology transfer with the group from South Africa The present assay format is functional at Saint-Justine Hospital and we have to be sure that all parameters of protocol are well defined and universal, so the assay can be reproduced, without technical help from the person who developed the protocol. Therefore, we will establish a technological transfer procedure and a corresponding manual, which will guarantee that the assay can be independently repeated with the same quality of assay performance as originally described. Here is the summary of current optimal conditions for each procedural step: (the details of protocol 4a to 4d might as well go to Appendix if you think it is a good idea) 4a) PCR The 50 l volume PCR was performed as originally suggested (van den Brule, et al., 2002) with minor modifications including shortening elongation and denaturation time to 20 seconds. Yield of PCR was monitoring by loading 10l reaction mix over agarose gel and EtBr staining. 4b) Conversion of PCR product to single stranded (ss) DNA and labeling The 40L were digested for 15 minutes at 37C, with EXO1 (NEB), following enzyme inactivation (20min, 85C), and lambda exonuclease (15 min, 37C, following 20 min, 85C). Sample was passed over S25 column for buffer exchange reaction compatibility with downstream T4 Polynucleotide Kinase labeling step. The conversion of double stranded to single stranded form of PCR product was monitored by disappearance of EtBr stained bend before and after digestion. 4c) Membranes Streptavidine-coated Promega membranes (SAM, Biotin Capture Membrane, Medison, WI) were used in the following manner. The 1 l of 100pmols/l of type specific (CP) 5’ biotinilated oligonculeotide probes were manually sported (HPV6, HPV11, HPV 16 and HPV 18) on the surface of 3cm x 2cm membrane (see Figure 1 for spotting schematic). The HPV universal probes (UP) were spotted using 1 l of 0.3 pmols/l of oligonucleotide. Spotted drops were dried at ambient temperature for 5 minutes, membrane was washed in ddH2O for 1-2 minutes and pre-hybridized in 2 mL hybridization buffer SSPE (150 mM NaCl, 10 mM NaH2PO, 1.1 mMEDTA, pH 7.4), 0.75MNaCl, 70mMTris–HCl, pH 7.4) containing 1% SDS and 200 mg/ml heparin (hybridization oven, Model 400, Robbins Scientific ) for 1-12h at 55oC. These membranes were either stored at room temperature for couple of days, or immediately used for hybridization assay. 4d) Hybridization Labeling of 2-20 pmols of PCR and/or oligonucleotide was perfomed using 5 l of [32P] ATP (6000 Ci/ mmol) and 1 L of T4 Polynucleotide Kinase to a specific activity of 105 to 106 cpm/pmol, following produce manual recommendation (Invitorogen). Hybridization was carried out for 1-12 hours. The membranes were then washed with 1x SSPE containing 0.1% SDS for 10 min at room temperature and either exposed overnight at -80°C with intensifying screens, or exposed in Cyclone Storage Phosphore Screen (Perkin Elmer) for 10-30 minutes and read by Cyclones software (OptiQuant, version 4.00).
The overall goal of this project is to develop a functional HPV assay that will be implemented in a clinical diagnostic laboratory in Quebec and in South Africa. The specific objectives required to reach this goal are
The point by point summary given below refers to major issues needed for assay optimization as well as validation of sensitivity and specificity of assay with standardized and clinical samples.
Cloning and sequencing of HPV PCR products will be performed in the case of discordant results. Activities which are currently performed to reach each objective are described in the following sections.
As presented in section 6.1, the actual prototype kit is composed of 4 probes designed to detect 4 HPV vaccine- relevant types (6, 11, 16 and 18) including two positive controls, one reflecting presence of any HPV in the sample and second, controlling for sample DNA integrity. HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived through amplification or chemically synthesized. We use of in vitro “reconstructed clinical samples” of HPV 6, 11, 16 and 18 following suggestion of Word Health organization for optimizing HPV tests. The variable input of GP5+/6+ HPV oligonucleotides or HPV plasmids is spiked prior to PCR with a constant amount of human DNA, originating from HPV negative cervical samples, to mimic molecular complexity of biological samples. In particular, range of genome equivalents (GE) of HPV (1000-10000) is added to human DNA. The concentration of human genomic DNA in reconstructed samples is comparable to the amount of DNA that is generally found in cervical scrape specimens (~106 human genomes/ml), (Quint et al., 2006). These “sample-reconstruction” experiments are allowing simulation of single and double HPV infection and controllable measure of analytical performance of our type-specific probes. In the next step, clinical samples (n=40) with known HPV status, as confirmed by the CHUM HPV assay (Fig. 9), will be used for estimating the performance of HPV assay. The results of HPV typing obtained with our probes and custom approach will be compared with the results of Roche LA HPV Genotyping Test. 
Figure 9. Schematic presentation of the CHUM HPV assay (a custom PCR-RFLP used for HPV typing)
There are multiple formats of multiplex hybridization kits on the market and custom assays in research or clinical laboratories. Several options for commercial format exist for our technology including solid support for DNA probe attachement and for hybridization signal detection. As previously stated, our prototype assay cannot reach the market in its current format, with its large SAM membrane, radioactive detection (P32), and high cost of production. As presented in Table 4, our rough estimation of the cost of production per kit is $26 which is highly expensive for such a kit. It should be noted that our kits have actually been manufactured in the researcher’s laboratory and there is no economy of scale. The membrane cost is clearly the main issue if we want to substantially reduce production cost.
Table 4. Cost breakdown for the current production of our HPV prototype assay Our data obtained so far shows that other membrane-based hybridization (Immunodyne ABC membrane, Pall) could replace SAM membranes (Promega). Preliminary data showing performance using modified streptavidin coated Immunodyne ABC membranes are given in Fig. 10. This might be an excellent alternative for reducing the cost of the final assay given the 100 fold lower price of Immunodyne ABC versus SAM membranes. 
Figure 10. Example of possible reduction of final product prize (100x) using Immunodyne ABC-Pall instead SAM-Promega, Immunodyne membrane can replace SAM (Promega) membranes used in the experiments shown in previous slides reducing thus the most significant material cost by 100 times (20$ to 0.2$) Development of an alternative system of visualization of probe–target hybridization is also essential for the simplicity and commercialization of the assay. One of the possibilities is a colorimetric assay with the use of 6-FAM labeled primers to produce PCR and a corresponding anti-6FAM-antibody with downstream alkaline phosphate-coupled colorimetric detection. Another important aspect will be to reduce the size of the assay. A 10X reduction of hybridization volume and corresponding solid support surface (beads, or array) will reduce PCR yield requirement to 50-100ng, typical for PCR volumes of 10uL. Detection of hybridization will be adequately adjusted. For example, the Luminex platform would require Cy3 fluorophore, while a slide micro-array format would allow use of DIG (digoxigenin-modified nucleic acids), or 6-FAM and the corresponding horseradish peroxidase-conjugated anti-fluorescein, or anti-DIG antibodies (Roche, Invitrogen and number of other suppliers). It is worthwhile mentioning that gold-nanoparticles are the simplest alternative for colorimetric detection of DNA (post-PCR) with robust chemistry and existing expertise in Canada (Yingfu Li, Department of Chemistry and Department of Biochemistry and Biomedical Sciences, School of Biomedical Engineering, McMaster University). In order to define the best format for the assay for commercialization, a deep analysis of the available options will be done. We plan to outsource this activity to a consultant with strong expertise in molecular diagnostics. This consultant has yet to be identified, but one possible candidate is Dr Yvan Côté, VP at Warnex, who has not only industrial experience but also who has expressed his interest in performing this analysis as a private consultant (independently from Warnex). It is worthwhile to mention that Yvan Coté has suggested that Warnex provides support to help in the design of the final format of the assay (see letter of support). Briefly, the analysis will have to take into account the scientific aspects (specificity & sensitivity), the commercial aspects (cost, license from third parties, others), as well as the regulatory requirement for the commercialization of the assay in Canada and South Africa. At least three meetings will be held with a consultant over the 8 week mandate. Participating in the meeting will be the inventors, Anne-Marie Larose from Univalor, and one representative from our potential commercial partners (Continental Diagnostic and Warnex). Meetings will include:
Experimental testing of the new format design will be performed before the beginning of the second phase of the project.
The objective to get clinical validation of our HPV assay, in a commercial format, is to conclude licensing agreement with commercial partners. It is clearly not the scope of this project to get regulatory approvals in Canada or South Africa. However, we consider it is important to establish what the regulatory requirements to reach the market are. These aspects have to be taken in account for the final design of the assay and in the licensing negotiation (will help both parties to better define license milestones). This task will be driven by Anne-Marie Larose from Univalor.
The outcome of phase 1 (first 20 weeks, i.e. five months) will define the choice of technological platform. Sensitivity and specificity of the assay will be re-assessed on the new assay format. This optimization step will required the following:
Since we anticipate a 10X reduction of hybridization volume and corresponding solid support surface (beads or array) this will allow reduction of total PCR yield (50-100ng), typical to the volume of 10uL. At this phase of the project, technique for visualization of the hybridization signal will be adjusted to best serve the chosen platform. Comparison with results obtained with the prototype assay will help the development and optimization of the new format of the assay. Optimization will be performed with the in vitro reconstructed sample, before testing clinical samples (see section 6.5.5). Some testing will also be conducted in at least one external laboratory, ideally by one of our commercial partners, in order to validate the reproducibility of new protocols. These experiments are not covered by this proposal and will be performed at the company’s expenses.
The performance (sensitivity and specificity) of the probes using the new platform will be compared with the typing results obtained in the first phase of the project. More precisely, the commercial format of the test will be used for HPV typing of the same 40 samples tested with our prototype assay, as described in section 6.5.1. Additional 100 clinical samples that are already charactrized by commercial tests like Roche or Hybrid Capture will be tested in this phase. The samples will be obtained through collaboration with Dr Francis Coutlée, Molecular Virology Laboraty at the Research Center of CHUM (see letter) who were running and partipating in variety of projects addressing biology, epidemilogy and diagnostics of HPV. The concordance analysis will follow.
The maturation project will require 12 months for its completion. As illustrated in table 5 the activities to meet the first objectives will start immediately while the activities for the second and third objectives will be performed concomitantly at the end of phase 1, most likely from week 10 to week 32. The activities for the last two objectives will be done successively. Dr Damian Labuda, Ms Sylvie Cossette and Ms Anne-Marie Larose, respectively the principal investigator with the inventors, the project manager and the representative of the partner organization, will meet on a regular basis, every 3 months to evaluate the progress of the project and the achievement of the milestones. The same committee will meet if necessary to evaluate any project of disclosure generated within the framework of this project in order not to reveal information that could preclude the filling of patent application.
Table 5: Gantt Chart for work plan
At the end of phase 1 we should have reach the first 3 objectives:
More specifically this means that we will get specificity and sensitivity data with clinical samples with the prototype assay and obtain the following results:
The design of the final format of the commercial product will need to be defined as well as the main regulatory requirement for the commercialization of the HPV kit. We will also need to get recommendations for the final design of the project. At the end of the second and last phases of the project we should have reach the 4th and 5th objectives:
Implementation of the new assay format in at least one clinical laboratory in Quebec, or South Africa will be performed.
At the end of phase 1, the project will be pursued only if we get positive results on clinical samples with the current prototype (SAM membrane and P32). Herein, clinical sample will have to give yield of GP56 PCR product in the minimal range of 100-1000ng (150nt long), which is in fact 1-10 pmols in total, the quantity typically detected in our present developmental experiments. This is, according to our observation and literature data, an average PCR yield of 50uL reaction volume of “classical” GP56 PCR after 40-45 cycles. We know that present solution for GP56 primers perform well for majority of HPV types, using 39 artificial targets. If our hybridization assay does not work in this range of concentrations we will consider that there is a significant failure in the process which will justify first NO GO.
Table 6. Proposed distribution of granting
In 2004, Ivan Brukner (see CV in Annex 3) joined our team to develop the iterative technology in its practical application focusing on the small sequence segments. His knowledge in nucleic acid chemistry matched the expertise of other members of the team in the physical chemistry of nucleic acids, in diagnostic application, in the development of genotyping tools, in the genetic epidemiology and pharmacogenetics, and importantly in the HPV DNA testing and in the epidemiology of HPV infections. Damian Labuda (see CV in Annex 3) has considerable experience in physical chemistry of nucleic acids, in genetic diagnostics, genetic epidemiology and in vitro selection. Ivan Brukner is the principal inventor of the proposed technology; he will be responsible for the technology implementation and daily follow-up of the experiments. Maja Krajinovic (see CV in Annex 3) has experience with the HPV DNA analysis, as well as in genetic epidemiology and pharmacogenetics; she will be involved in design of reconstructed sample experiments, selection of clinical samples (with Izabella Gorska-Flipot) and between tests concordance analysis. Izabella Gorska-Flipot (see CV in Annex 3), at the Hospital Hotel-Dieu, has a longstanding experience in the molecular diagnostics and in the HPV cervical infections in particular; she will be responsible for the analysis of clinical samples using custom approach and commercial kit. Our achievements are summarized in 4 published manuscripts Nucleic Acids Research, 2007; Journal of Clinical Virology, 2007; Nature Protocols, 2007 and Int. J Cancer., 2008. In addition, intellectual property related to our technology is protected. The present application is to finalize the prototype of the HPV diagnostic device and to validate its use in the clinical setting.
Gestionnaire de projet au Centre hospitalier universitaire Sainte-Justine (CHU Sainte-Justine) Le CHU Sainte-Justine est un des plus grands centres hospitaliers universitaires pédiatriques du Canada. Il a pour mission d’améliorer la santé des enfants, des adolescents et des mères du Québec. Afin d’arriver à assumer pleinement sa mission et surtout son rôle en tant que centre universitaire d’enseignement et de recherche, le CHU Sainte-Justine compte sur l’excellence de la recherche de ses chercheurs regroupés dans son Centre de recherche. Au cours des dix dernières années, le Centre de recherche du CHU Sainte-Justine a connu une croissance sans égal parmi les dix-neuf centres et instituts de recherche subventionnés par le FRSQ. Depuis sa fondation en 1973, le Centre de recherche s'est transformé en un réseau d'axes de recherche pluridisciplinaire allant de la recherche biomédicale à la recherche clinique en passant par la recherche sur les soins et le système de santé, la santé des populations et l'évaluation des technologies. Le leadership du CHU Sainte-Justine en recherche est aussi fondé sur un partenariat actif et engagé dans des réseaux de recherche. Depuis 2003, 30 nouveaux chercheurs ont été recrutés portant le nombre de chercheurs à temps complet à 90. La progression du nombre d'étudiants qui est passé de 276 à 402 au cours des cinq dernières années témoignent du pouvoir attractif du Centre, de la qualité de ses chercheurs et de sa mission de former du personnel hautement qualifié au niveau académique de même que pour l'industrie biomédicale et pharmaceutique. Les publications témoignent de notre productivité scientifique comme centre de recherche. De 2004-2005 à 2007-2008, le nombre d'articles avec comité de pairs a augmenté annuellement de façon constante passant de 364 à 385. Parallèlement à cette productivité scientifique du Centre de recherche, le CHU Sainte-Justine aura été le foyer d'une importante activité en valorisation de la recherche et des connaissances dans le domaine de la santé. Au cours des cinq dernières années, plus d'une trentaine de chercheurs ont soumis une soixantaine de déclarations d'inventions menant à trente-deux demandes de brevets, aboutissant à une dizaine d'accords commerciaux, dont neuf licences et la création d'une entreprise dérivée. Également, le Centre de recherche a assisté à une croissance continue des contrats de recherche en partenariat avec l'industrie et autres centres d’enseignement, totalisant plus de 100 nouveaux contrats en 2006 dont la valeur atteint plus de 22 millions de dollars, témoignage tangible de la valorisation du savoir-faire et connaissance de la communauté des chercheurs du CHU Sainte-Justine. Le Centre de recherche, sous la direction du Dr Guy A. Rouleau depuis près de trois ans s’est doté d’une structure administrative qui facilite le support à la recherche et la valorisation des résultats. Sous la direction de Madame Sylvie Cossette CA, adjointe au directeur et comptant plus de 15 ans d’expérience comme gestionnaire au Centre de recherche, deux professionnels spécialisées en ententes de recherche et en valorisation sont à la disposition des chercheurs. De plus, un chercheur clinicien est délégué par le Centre de recherche pour siéger sur le comité d’évaluation des technologies de sa société de valorisation Univalor. Madame Cossette sera la gestionnaire de projet de maturation technologique du CHU Sainte-Justine et sera responsable de s’assurer de son bon déroulement. Elle devra notamment :
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||