Patent Application
20110183313
The present invention relates to the identification of human host factors involved in the early stage of HIV infection. Furthermore, it relates to the use of the identified genes for the elucidation of the mechanism of HIV-infection, as drug targets, and for identifying a compound useful in the treatment of HIV.
A majority of chronic diseases and infection manifest at the integrated level of the cell. Examining disease progression by live cell imaging allows for a high degree of resolution, with the visualization of molecular disease mechanisms and their response to genetic changes. While this type of approach has been very successful in individual experiments, it has remained largely refractory to systematic, genome wide analyses.
During evolution HIV has learned to subcontract a large part of its life cycle to human host factors. In the last decade, several such host factors have been identified through a multiplicity of approaches (for review see1). Few of the approaches in the identification of host factors have been systematic, and so far none has been exhaustive. However, the availability of host factors is not only important for the full understanding of the HIV life cycle. Effective ways to mine for viral host factors would further allow the comparison of factor specificity of HIV subtypes with each other or with SW (Simian immunodeficiency virus), thus pointing to crucial mechanisms in the manifestation of infections, and potential therapy targets.
It was an object of the present invention to identify human host factors involved in the early stage of HIV infection for use in the elucidation of the mechanism of HIV-infection, as drug targets, and for identifying a compound useful in the treatment of HIV.
The objects of the present invention are solved by a nucleic acid having a sequence represented by SEQ ID No. 46, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, for use
a) in the elucidation of the mechanism of HIV-infection;
b) as a drug target; or
c) in the identification of a compound useful in the treatment of HIV.
The objects of the present invention are also solved by a nucleic acid having a sequence represented by SEQ ID No. 1 or SEQ ID No. 2, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, for use
a) in the elucidation of the mechanism of HIV-infection;
b) as a drug target; or
c) in the identification of a compound useful in the treatment of HIV.
The objects of the present invention are also solved by a nucleic acid having a sequence represented by SEQ ID No. 5, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, for use
a) in the elucidation of the mechanism of HIV-infection;
b) as a drug target; or
c) in the identification of a compound useful in the treatment of HIV.
The objects of the present invention are also solved by a nucleic acid having a sequence represented by any of SEQ ID Nos. 3-4 and 6-45, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, for use
a) in the elucidation of the mechanism of HIV-infection;
b) as a drug target; or
c) in the identification of a compound useful in the treatment of HIV.
The term “nucleic acid” as used herein is meant to refer to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
The term “sequences complementary to said nucleic acid or to said partial sequences” as used herein also refers to the corresponding (complementary) RNA sequences or partial sequences.
The minimum length of 20 contiguous nucleotides of said partial sequences ensures their specificity. In a preferred embodiment, said partial sequences comprise at least 21, in an even more preferred embodiment at least 22 contiguous nucleotides.
The term “drug” as used herein is meant to refer to a pharmaceutical agent that is suitable for the treatment of HIV.
The initial identification of a compound inhibiting (a) binding to, (b) activity of, or (c) expression of a target protein can be achieved experimentally or be based on available information concerning the target. Compounds inhibiting (a) and (b) are active on the protein level. Compounds inhibiting (c) are directed at the nucleic acids (e.g. DNA, RNA, mRNA) encoding the protein or having a regulatory function. The ability of a compound to bind to a protein can be determined using techniques such as competitive and non-competitive binding assays. Such assays can be performed, for example, using a labeled compound (direct measurement) or detectable reagents that bind to the respective compound (indirect measurement). The encoding nucleic acid sequence of an identified target protein (such as a gene/nucleic acid represented by SEQ ID Nos. 1 to 46, preferably SEQ ID No. 46, 1-2 or 5) provides a target for compounds that are able to hybridize to the nucleic acid. Examples for such compounds include siRNAs, ribozymes, and antisense nucleic acids.
Preferably, a nucleic acid having a sequence represented by any of SEQ ID Nos. 1 to 46, preferably SEQ ID No. 46, 1-2 or 5, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, are used as a drug target or in the identification of a compound useful in the treatment of HIV according to the methods outlined in WO 2008/03462228.
Preferably, a method of identifying a small molecule modulator (which also represents a compound useful in the treatment of HIV) for a target protein candidate (a protein encoded by any of SEQ ID Nos. 1 to 46, preferably SEQ ID No. 46, 1-2 or 5) comprises the following steps:
Preferably, said first cell and said second cell of the same type as said first cell are LTR-GFP HeLa cells (LTR=long terminal repeat).
Preferably, a nucleic acid having a sequence represented by any of SEQ ID Nos. 1 to 46, preferably SEQ ID No. 46, 1-2 or 5, or partial sequences thereof, or sequences complementary to said nucleic acid or to said partial sequences, said partial sequences comprising at least 20 contiguous nucleotides, are used in the elucidation of the mechanism of HIV-infection by utilizing standard methods in the field of network or systems biology, including but not limited to proteomic interaction maps, pull-down experiments (i.e. AP/MS), microarray analysis, and computational modeling29.
SEQ ID Nos. 1 to 46 are listed in Table 2.
Through a combination of genome wide RNA interference, high-density cellular micro-arrays, confocal imaging and image analysis, the inventors have identified so far unknown human host factors involved in the early stage of HIV infection. Using the HIV receptor molecule CD4 as a classifier on 7 visual genome wide analyses, they have shown that HIV-1 employs 0.21% of the human genome, or 44 host genes, to complete its early live cycle. By combining the high information content of visual images with the rapid reproducibility of array-based genome wide screens, the present approach allowed the identification of HIV-1 host factors with high reliability
To directly identify genes involved in HIV/disease pathogenesis at a cellular level, the inventors have developed a genome wide visual RNA interference approach. It was designed as an automated analysis of pathway and pathogen activity within living cells, using quantitative imaging tools2. Genome wide libraries of short interfering RNAs (siRNAs) and short hairpin RNAs in viral vectors have been screened with low resolution in microtiter plates at a one well per gene level3. Extending this to high resolution imaging has proven to be very challenging. Cellular microarrays are an alternative, versatile solution for imaging cellular events4,5. Cell monolayers overlaid on printed spot arrays of siRNA, viral particles or compounds that reverse transfect the cells4-10 provide an alternative to laborious robotic screening4,7,8,10-12.
The inventors have exploited these advantages to screen HIV infection using cellular microarrays.
Reference is now made to the figures, wherein
In
Cellular microarrays were produced to cover the human genome in a minimal number of arrays, permitting confocal imaging, and removing any dependence on mechanical accuracy for siRNA spot location and imaging7. Arrays printed onto 24×60 mm optical glass wafers were mass produced using a high throughput contact printer. Individually bar-coded arrays comprised 3,888 siRNAs as 300 μm diameter spots at a pitch of 500 μm in 108 columns×36 rows. siRNAs were encapsulated in a mixture containing transfection reagent and gelatin7-9,12,13 and red fluorescent siRNA. Red fluorescent siRNA gave optically identifiable individually addressable spots and the entire array could be visualized after cell overlay (
The HIV infection assay comprised 28 hours reverse transfection of HeLa LTR-GFP cells followed by 48 hours of infection with an HIV-1 MOI of 0.14. HIV infection enabled TAT driven expression of the stably integrated GFP and thus recapitulated early steps in viral infection14. Under these conditions, HIV infection was significantly repressed in cells transfected with a CD4 siRNA (
A collection of 84,508 siRNAs, corresponding to four unique siRNA duplexes targeting each of 21,127 unique human genes with control siRNAs, in an encapsulation mixture were printed. Each array comprised 3,888 spots, including 648 controls and the entire human genome was covered in 7 slides (
A single visual genome wide analysis for HIV infection is shown in
A first image analysis goal was to identify and annotate spots on the arrays. Here, there is no relationship between the printed array, siRNA annotation and image sequence (
Seven complete human genomes, a total of 49 siRNA arrays, were cultivated for 28 hours to permit host gene silencing. Arrays were infected with live HIV-1 at a multiplicity of infection of 0.14 for 3 hours, washed and incubated for 45 h prior to imaging. Once the grids were fitted and the identity of each spot retrieved, HIV infection was independently analyzed on each spot using the following image and data analysis strategy. Measurement of GFP fluorescence alone would yield no information on cell shape, density and cell:cell fusion. More importantly, dilution of the GFP signal during syncitial formation can not easily be distinguished from repression of GFP production. To address this, the inventors developed an algorithm that retrieved 15 descriptors.
To be able to quantify HIV infection, the inventors measured cells within the siRNA spot and in a border around the spot. This was achieved using an algorithm that randomly measured pixels in an image greater than the spot to define the best possible spot location (
Given the variations in cell culture, the measurements of infection were normalized across the arrays. The array is a large experiment with a low frequency spatial variation due to imperfect cell density across its surface. Array images representing each dimension of the result vector for a whole single array are shown in
The inventors' intent was to identify genes that were as potent in repressing early stage HIV infection as the control CD4. Thus, they built a two class classifier from two control distributions of 215 individual CD4 and 3896 SCRAMBLED experiments. Since they were both 15-variate Gaussian distributions, they set a simple and robust classifier and computed the most discriminating projection as shown in
Of these 44 genes, 36 were identified as novel in terms of their involvement in HIV infection (see Table 2). The remaining eight genes have been previously shown, either directly or indirectly, to be involved in HIV infection. Recently, a functional genomic screen by Brass et al.18 revealed MED28, CSPP1 and ERP27 as three of several host genes which encode for proteins required for HIV infection. Prior to this, MED28 (magicin) had already been identified as an interacting partner of FYN19, a known HIV interacting partner. Ku70 is a well known mediator of the early steps of retroviral infection due to its interaction with retroviral replication intermediates and pre-integration complexes20. PKN1 (also known as Pak1) has been shown to interact with the HIV accessory factor Nef, and depletion of PKN1 strongly inhibited HIV infection in multiple cell systems21. The FDA-approved drug phenylbutazone, which targets PTGIS, has been patented as a potential antiviral (including HIV) agent for both humans and animals22. CCL2/MCP-1 codes for a proinflammatory chemokine which is induced by the HIV matrix protein p17 during HIV infection23, 24. Furthermore, it has previously been reported that UNG2 is packaged into HIV viral particles and physically associates with the viral reverse transcriptase enzyme25. The fact that over 20% of the identified genes are known effectors of HIV infection validates the overall results presented herein. Interestingly, mutations in the RNASEH2A gene have been shown to result in the neurological disorder Aicardi-Goutières syndrome (AGS)26.
To verify the significance of the screening results, it is essential to prove that, ultimately, depletion of the candidate genes in LTR-GFP HeLa cell blocks HIV replication in a way similar to that seen by CD4 knockdown. To test this, we selected RNASEH2A, MED28 and JMY and used CD4 as a control. Cells were transfected with individual siRNA for 24 hours and were infected with HIV for 48 hours. Viral replication was measured by following the appearance of GFP and P24 expressing cells.14 RNASEH2A, MED28 and JMY knockdown in cells blocked HIV infection (
To further verify the significance of the screening results, RNASEH2A was used as a representative gene for knockdown in a more representative cell type for HIV infection. For effective gene silencing, individual RNASEH2A #1 and #2 siRNAs were transfected into Jurkat (T lymphocyte) cells. It was verified that the targeted mRNA was down-regulated by its expression by RT-PCR both in a qualitative (
All fine chemicals were purchased from Sigma-Aldrich. DRAQ5 was from BioStatus (Shepshed, UK). All siRNA duplexes were purchased from Dharmacon (USA). The siRNA library comprised 1.0 nM of the Dharmacon siARRAY whole human genome siRNA library (Thermofisher, West Lafayette, Colo.) containing to 84,508 siRNAs corresponding to four unique siRNA duplexes, targeting each of 21,127 unique human genes. Primary antibodies were from Santa Cruz Biotechnology, and all fluorescent secondary antibodies were from Molecular Probes/Invitrogen (Carlsbad, Calif.). Transfection reagents were from commercial sources.
LTR-GFP HeLa cells (A. Boese, Institut Pasteur Korea) were produced as described14 wild type HeLa (ATCC) and GFP-torsin expressing HeLa (R. Grailhe, Institut Pasteur Korea) were cultivated in high glucose glutamax Dulbecco's modified eagles medium (Invitrogen; Carlsbad, USA) supplemented with 110 mg/mL sodium pyruvate, 10% fetal calf serum (Gibco, USA) and 1% penicillin streptomycin (Invitrogen; Carlsbad, USA). Stable lines were maintained in medium supplemented with selection marker. Cell lines were cultivated on arrays for 12 to 72 hours for quantifying reverse transfection. For HIV infection, 650,000 cells were seeded per array (24×60 mm) and cultivated in Opti-MEM (Invitrogen; Carlsbad, USA) supplemented with 5% fetal calf serum (Gibco, USA) and 1% penicillin streptomycin (Invitrogen; Carlsbad, USA) for 28 hours. Cells were inoculated overnight with HIV-1 strain IIIB (Daymoon industries; Cerritos, USA) at an MOI of 0.14. Fresh Opti-MEM (Invitrogen; Carlsbad, USA) supplemented with 5% fetal calf serum (Gibco, USA) and 1% penicillin streptomycin (Invitrogen; Carlsbad, USA) was added the following day. Cells were cultivated for an additional 45 hours, followed by fixation in 1% (w/v) paraformaldehyde in Dulbecco's phosphate buffered saline, and nuclei were stained with 2.5 μM Draq5 (BioStatus, UK) before imaging. Jurkat clone E6-1 (ATTCC) was cultured in RPMI medium 1640 (Invitrogen; Carlsbad, USA) supplemented with 10% Fetal Calf Serum (Gibco, USA), 1% penicillin streptomycin (Invitrogen; Carlsbad, USA), 1 mM sodium pyruvate (Gibco, USA), 10 mM HEPES.
siRNA transfection solution was prepared essentially as described27 and printed as 3,888 spot arrays (108×36 spots) on No. 1 glass coverslips using stealth pins (telechem, USA) and a high throughput microarray printer (Genomic Solutions, USA) at 22-25° C., 55-65% RH enclosed in a custom built clean chamber providing a sterile HEPA filtered atmosphere. Arrays were stored in a dessicator with no significant alternations in performance from 1 week to 8 months post-printing. 7 slides covered the genome and contained 16% of control siRNA spots.
Arrays were acquired with a point scanning confocal reader (Imageexpress Ultra, Molecular Devices, USA) as 16 bit TIFF files written directly to an external database. Images were read directly from the database for analysis using software designed for this purpose. Adaptive gridfitting was applied to identify siRNA spots in the entire array, fit the spots and crop them before extracting the image data for analysis, annotation and result export.
Total RNA was isolated from siRNA transfected LTR-GFP HeLa and/or Jurkat cells by Trizol method (Invitrogen, USA). cDNA was made using 1 μg of total RNA and MMLV-reverse-transcriptase (Promega) in a 25 μl reaction mixture in the presence of 50 pmol oligo(dT) primer and 20 μM dNTP mixture for 60 mM at 37° C. For PCR amplification, specific oligonucleotide primer pairs (0.2 μmol each) were incubated with 200 ng cDNA, 1 unit of LA Tag polymerase (Takara), 1×LA PCR buffer 2 (2.5 mM MgCl2) and 100 μM dNTP in a 25 μL reaction mixture. The sequences of primers used are as follows:
PCR conditions were 95° C. for 30 sec, 54° C. for 30 sec, and 72° C. for 3 mM, for a total of 40 cycles. The PCR products were applied onto a 1% agarose gel and visualized with Ethidium bromide.
Forward siRNA Transfection and Virus Infection
Jurkat cells (40,000 cells/well) were transfected with 1 μM Acell siRNA (Dharmacon, USA) against selected individual RNASEH2A#1, #2 or scrambled in 24-well plates, and then incubated for 72 hours. Cells were infected with HIV-1, Strain IIIB virus (Daymoon industries; Cerritos, USA) MOI 0.5, 0.01 and inoculated for 3 hours. After viral supernatant was removed, cells were cultivated in RPMI medium 1640 (Invitrogen; Carlsbad, USA) supplemented with 10% Fetal Calf Serum (Gibco, USA), 1% penicillin streptomycin (Invitrogen; Carlsbad, USA), 1 mM sodium pyruvate (Gibco, USA) and 10 mM HEPES for 96 hours. Virus replication was determined by detection of p24 HIV-1 viral core antigen in cell-free supernatants by a P24 ELISA (Perkin-Elmer).
LTR-GFP HeLa cells (5,000 cells/well) were transfected with 50 nM of a selected siRNA (Dharmacon, USA) in 96-well plates followed by incubation for 24 hours. Cells were infected with HIV-1, Strain IIIB virus (Daymoon industries; Cerritos, USA) and inoculated for 3 hours. After viral supernatant was removed, cells were cultivated in growth medium. Cells were fixed in 4% (w/v) paraformaldehyde in Dulbecco's phosphate buffered saline, stained with Anti-P24 antibody (Abcam, USA) and stained with 2.5 μM Draq5 (Biostatus, UK) before imaging.
Cells were washed twice with phosphate-buffered saline (PBS), fixed for 10 min with 4% (w/v) paraformaldehyde in PBS and then washed with PBS. For permeabilization, cells were incubated in 0.1% Triton-X 100 in PBS for 10 min and subsequently washed in PBS. Then followed incubation with a 1:200 dilution of mouse anti-P24 antibody in 10% goat serum in PBS for 2 hours at 4° C. Plates were washed 3 times with PBS for 10 min on an orbital rotator. Alexa 532 goat anti-mouse secondary antibody (1:1000) was incubated with the cells for 1 hour at room temperature and cells were then washed 3 times for 10 min with PBS on an orbital shaker before the addition of 5 μM of DraQ5 in PBS for 10 min at room temperature.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP09/04604 | 6/25/2009 | WO | 00 | 3/18/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61133027 | Jun 2008 | US |
