GB2448994A

GB2448994A - shRNA sequences

Info

Publication number: GB2448994A
Application number: GB0808017A
Authority: GB
Inventors: Ralph Brandt
Original assignee: Galapagos NV
Current assignee: Galapagos NV
Priority date: 2007-05-04
Filing date: 2008-05-02
Publication date: 2008-11-05
Also published as: GB0708662D0; GB0808017D0

Abstract

Short hairpin RNA (shRNA) polynucleotides comprising an RNA sequence comprising a first stretch of 19 consecutive nucleotides and a second stretch of 19 consecutive nucleotides, which is the reverse-complement of the first stretch, wherein in said second stretch: a) in the 5'-3' direction, the last nucleotide is a C nucleotide, b) no stretches of four or more consecutive identical nucleotides are present, c) G+C is 30-70% and d) in the 5'-3' direction, consecutive nucleotides 1-19 are homologous to an RNA molecule. Also claimed are vectors comprising such molecules, and methods of preparing such vectors.

Description

shRNA sequences

Background of the Invention

The present invention relates to polynucleotide constructs, methods for their preparation, and preparations for their use in methods that lower the amount of RNA and/or protein production in cells based on the intracellular expression of small hairpin polyribonucleic acid (5hRNA) molecules.

Pharmaceutical companies are interested in reliable knockdown based technologies as many drug screens for small molecules are based on inhibiting the activity and effect of an expressed protein. Therefore, blocking the expression or function of a potential target, either through screening in a cellular assay or through single gene validation will provide an important data set regarding the disease modifying role of the target early on in the drug development process.

This data set forms a strong basis for the start of a drug development program, based on a compound, antibody or biological, with the aim to develop an effective therapy.

Various knockdown or knockout approaches are used to study gene function in mammalian cells (e.g. antisense, antibodies, ribozymes, aptamers, zinc finger proteins, chimeric RNA-DNA oligos, etc.). However, these technologies are not robust and efficient nor can they be generically applied to all genes and all cell types.

RNA interference (RNAi) is the post-transcriptional process of gene silencing mediated by double stranded RNA (d5RNA) that is homologous in sequence to the silenced RNA and is observed in animals and plants. The dsRNA is processed into 21-23 nucleotides (nts) molecules, called small interfering RNAs (siRNAs), which guide the sequence-specific degradation of the target RNA.

RNAi provides researchers with an additional genetic tool to study gene functions. In C. elegans, chromosomes I and III have now systematically been analyzed for phenotypic effects. The RNAi approach also creates extra possibilities in developmental studies. Classical knockouts with lethal effects during development could never be analyzed in later developmental stages. With RNAi, the onset of the effect may be varied and roles in later stages of development may be studied.

The use of RNAI in mammalian cells has been problematic since introduction of long dsRNA (>30 base pairs) results in two major intracellular responses; activation of the double stranded RNA dependent protein kinase PKR, which results in a general block of protein synthesis and activation of the interferon-induced (2'-5') oligoadenylate synthetase. Upon activation, this enzyme polymerizes AlP into 2'-S'linked nucleotide oligomers (also indicated by 2-5A). The 2- 5A oligomers activate the ribonuclease RNase L that results in RNA degradation.

Recently, it has been demonstrated that RNAi can be used in a panel of mammalian cell lines.

The approach is based on direct transfection of the 21-23 nts siRNA duplexes into the cells. This circumvents the intracellular responses mentioned above and results in sequence-specific silencing of endogenous and heterologous genes.

An important bottleneck in the s1RNA transfection approach is its limited applicability to target different cell types, especially primary cells. Primary cells are closest to the in v/ye situation and often have the highest physiological relevance. Non-viral DNA or 5iRNA transfection technologies have severe limitations with regard to these cells and are not efficient and reliable.

Practical use of these approaches needs significant optimization of conditions, and in general lacks the robustness necessary for large-scale applications. Furthermore, the gene transfer reagents used are often toxic, yielding lower levels of viable transduced cells. In essence, they do not allow a generic 51RNA application for a wide variety of cell types, including primary cell types such as T cells, B cells, mast cells, endothelial cells, synoviocytes and lung epithelial cells.

Furthermore, transfection of the siRNA gives a short knock-down effect. For a prolonged knock-down effect in cells several additional transfections are necessary.

Viral constructs encoding the 5iRNA molecule circumvent the problems described above. WO 03/020391 describes adenoviral vectors which express short hairpin RNAS (shRNAs) that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a functional knock-down of the encoded protein.

However, not every 5iRNA can effectively downregulate a gene. siRNAs directed to different regions of a mRNA can result in different levels of gene silencing. It is estimated that between 25% and 75% of siRNAs are effective and a high degree of variability is seen between specific RNAi constructs, some demonstrate a 10-fold effect, some demonstrate a 50-fold effect, whereas others do not appear to have any effect. For example, an 5iRNA directed against vimentin (nt 346-368 from Genbank (NCBI): NM003380 relative to start codon) did not give a silencing effect. Three other siRNAs against vimentin were designed and these all gave an effective gene silencing effect (nt 1145-1167, nt 863-885 and nt 1037-1059 from NM_003380 relative to start codon, (Elbashir et a!., 2001)). A procedure for designing siRNAs for efficiently inducing RNAi in mammalian cells has been suggested (Elbashir et 8/., 2002). However, an siRNA against c-myc, designed according to this protocol was ineffective in silencing. and did not show a reduction in protein expression (Jarvis and Ford, 2002). This clearly demonstrates that target site selection is critical for the effective induction of RNA1 by sIRNA5.

Summary of the Invention

In one aspect the present invention provides polynucleotides that are highly effective in silencing an RNA molecule.

In a further aspect the present invention provides vectors which comprise a nucleotide of the invention operatively linked to a promoter sequence.

The present invention also provides libraries of polynucleotides of the invention and methods for 1 0 their production.

Additionally, the present invention provides uses of the polynucleotides of the present invention.

Description of the Invention

The present invention provides polynucleotides that are highly effective in silencing an RNA molecule.

The present invention provides a polynucleotide comprising an RNA sequence comprising: a) a first stretch of 19 consecutive nucleotides, and b) a second stretch of 19 consecutive nucleotides, wherein said second stretch is the reverse-complement of the first stretch of 19 consecutive nucleotides, and wherein in said second stretch of 19 consecutive nucleotides: i). in the 5' -> 3' direction, the last nucleotide is a Cnucleotide; ii). no stretches of four or more consecutive identical nucleotides are present; iii). the total number of G-and C-nucleotides is between 30-70% of the total number of nucleotides; and iv). in the 5' -> 3' direction, consecutive nucleotides 1-19 are homologous to an RNA-molecu le.

The consecutive nucleotides 1-19 in the 5' -> 3' direction of the first stretch of 19 consecutive nucleotides are homologous to the RNA-molecule to be silenced.

In a further embodiment the invention provides polynucleotides wherein the RNA-molecule is a human RNA molecule. In a specific embodiment an RNA molecule in the target cell will be silenced by the polynucleotides of the present invention. In a further specific embodiment, the RNA molecule to be silenced is a drugable gene.

As used herein the term "drugable gene" refers to a gene with a pharmaceutical value, i.e. one that can be used to discover and develop small molecule drugs. New drugable genes can be found on the basis of their similarity to proteins which have proven amenable to small molecule compound development in the past. Known drugable gene classes include but are not limited to: G-protein coupled receptors (GPCR5), ion channels, nuclear hormone receptors, kinases, phosphatases, proteases and other enzymes.

In a further embodiment the invention provides polynucleotides wherein in the first stretch of 19 consecutive nucleotides no stretches of three or more consecutive A-nucleotides are present.

In a further embodiment the invention provides polynucleotides wherein in the first stretch of 19 consecutive nucleotides no stretches of three or more consecutive U-nucleotides are present In one embodiment the invention provides polynucleotides wherein, in the 5' -> 3' direction, consecutive nucleotides 1-19 of the first stretch of 19 consecutive nucleotides are a unique sequence. This unique oligonucleotide sequence is homologous to a stretch of 19 consecutive nucleotides that is found only once in known sequences, excluding transcript variants of the same gene. Sequence databases can be searched to identify sequences which occur only once.

Known databases include the EST database, the EMBL nucleotide sequence database, GenBank, and the Entrez nucleotide database, but a person of skill in the art will appreciate that there are alternative databases and the present invention envisions the use of any one of these to verify the unique status of the sequence. Where a polynucleotide contains a unique sequence corresponding to consecutive nucleotides 1-19 in the 5' -> 3' direction of the first stretch of 19 consecutive nucleotides, it will therefore silence only one specific mRNA molecule.

In an alternative embodiment, the sequence of consecutive nucleotides 1-19 in the 5' -> 3' direction, of the first stretch of 19 consecutive nucleotides may be found more than once in sequence databases. These polynucleotides may therefore be used to silence alternative gene transcripts of the same sequence or alternatively spliced variants. In addition, a person of skill in the art will be able to design the first stretch of 19 consecutive nucleotides such that the sequence is found in more than one member of a family of proteins. Such a nucleic acid sequence may then be used in accordance with the methods described in more detail herein to silence more than one member of a family of proteins.

According to another embodiment of the invention, polynucleotides are provided wherein, in the 5' -> 3' direction consecutive nucleotides 1-19 of the first stretch of 19 consecutive nucleotides are homologous to a sequence positioned at least 75 nucleotides downstream of the translation initiation site of the transcribed RNA molecule encoding a polypeptide.

According to a further embodiment of the invention polynucleotides are provided wherein, in the 5T -> 3' direction, consecutive nucleotides 1-19 the first stretch of 19 consecutive nucleotides are homologous to a sequence positioned at least 50 nucleotides upstream of the translation termination site of the transcribed RNA molecule encoding a polypeptide.

In another embodiment, the present invention provides polynucleotides, wherein the RNA sequence also comprises a linker sequence linking the first stretch of 19 consecutive nucleotides with the second stretch of 19 consecutive nucleotides.

In a further embodiment the linker sequence is 4-30 nucleotides long, particularly 5-15 nucleotides long and most particularly 12 nucleotides long.

In a specific embodiment the linker sequence is GUUUGCUAUAAC (SEQ ID NO: 7471).

In one embodiment the invention provides polynucleotides, wherein the first stretch of 19 consecutive nucleotides is selected from a group consisting of SEQ ID NO: 1-7470.

Another embodiment of the invention provides a vector capable of transfecting a host cell, wherein said vector comprises a sequence encoding a polynucleotide of the present invention and a promoter sequence operatively linked to the sequence encoding the polynucleotide.

In a specific embodiment, the promoter is a microRNA promoter, for example a let-7 promoter.

More particularly the promoter is a promoter recognized by RNA Polymerase III, for example the U6 small nuclear RNA promoter. A person skilled in the art will appreciate that other promoters recognized by Polymerase III may also be used in the vectors of the present invention such as, Hi, tRNA, 5nRNA, VA RNA and 5S rRNA.

According to a further embodiment of the invention, vectors are provided wherein the vector is an adenoviral vector, in a specific embodiment the adenoviral vector is replication defective.

The replication defective adenoviral vectors may be El-deleted or El and E2A deleted. The adenoviral vectors include the El-deleted adenoviral serotype 5 vectors. Vectors may also be prepared from other adenoviral serotypes and corresponding packaging cells that include sequences for viral proteins deleted from such vector backbones. A suitable approach of the present invention has an adenoviral vector/packaging cell wherein the packaging cell and vector do not include any overlapping adenoviral sequences, which overlap would provide the statistical possibility of the production of replication competent adenoviral particles. Packaging cells useful in the production of such vectors include the 293 and 911 cells, with the most suitable cells being the PER.C6 cell line. The modified PER.C6/E2A cell line is especially suitable. It complements the El, E2A deleted adenoviral vector constructs, with non-overlapping adenoviral El, E2A sequences. Other viral vector systems can be used such as the retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, M0MuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentivirus vector systems such as human immunodeficiency virus (HIV) or equine lentivirus may also be used in the practice of the present invention. Another suitable viral vector system is the adeno-associated virus ("AAV"). The MV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. It is also possible to introduce a DNA vector in viva as a naked DNA plasmid.

Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter.

The invention further provides libraries of polynucleotides of the present invention.

Preferably the library of the present invention provides vectors according to the present invention. The vectors may comprise plasmids, naked RNA. Preferably, the vectors are viral vectors preferably selected from a group consisting of MV, Lentivirus or Retrovirus.

Alternatively, more than one vector can be introduced into a single host cell, thereby introducing more than one stretch of 19 consecutive nucleotides.

More preferred vectors are adenoviral vectors, preferably the adenoviral vectors are replication defective. Replication deficient vectors may be multiplied in a packaging cell having complementary sequences to the sequence contained in the vector itself.

The present invention also provides means to prepare libraries of polynucleotides and vectors as described herein. These libraries may be prepared as a single element, as compartmentalized, or as discrete elements. Alternatively, a library comprising pools of vectors may be prepared.

Methods are described for making a vector comprising the synthesis of a forward primer and a reverse primer.

a. The forward primer is synthesized with the following sequence in the 5' -> 3' direction: the nucleotides ACC ii. the reverse-complement sequence corresponding to the DNA sequence from step a-iv iii. the nucleotides GTTTGCTATAAC (SEQ ID NO: 7472) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 v. the nucleotides T1T b. the reverse primer is synthesized with the following sequence in the 5' -> 3' direction the nucleotides TAAAAA ii. the reverse-complement sequence corresponding to the DNA sequence from step b-iv iii. the nucleotides G1TATAGCAAAC (SEQ ID NO: 7473) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 c. The primers are annealed and cloned in plasmid pKD122, thereby exchanging the ccdB sequences for the annealed primers.

The orientation of the sequence encoding the sequence selected from the group consisting of SEQ ID NO: 1-7470 can be reversed. The forward and reverse primers look then as follows: a. forward primer with the following sequence in the 5' -> 3' direction: the nucleotides ACC ii. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 iii. the nucleotides GT1TGCTATAAC (SEQ ID NO: 7472) iv. the reverse-complement sequence corresponding to the DNA sequence from step a-u v. the nucleotides TIT b. a reverse primer with the following sequence in the 5' -> 3' direction i. the nucleotides TAAAAA ii. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 iii. the nucleotides GTTATAGCAAAC (SEQ ID NO: 7473) iv. the reverse-complement sequence corresponding to the DNA sequence from step b-u The invention further provides a method of determining the function of a naturally occurring polynucleotide sequence, said method comprising: a) transfecting a host cell with a vector as described above, wherein the vector encodes/comprises a polynucleotide sequence of the invention, and b) detecting a change in cellular phenotype.

According to a specific embodiment, the present invention provides a method of determining the function of a naturally occurring polynucleotide sequence in a high throughput setting, said method comprising: a) providing a library of vectors b) transducing a host cell with the vectors of step (a), c) expressing in the host cell the product(s) of the vectors of step (a), d) thereby altering a phenotype of the host, e) identifying the altered phenotype and, f) assigning a function to the naturally occurring polynucleotide sequence (s).

The libraries according to the invention may be used to assist in the elucidation of the functions of host cell RNA molecules including the polynucleotide of the present invention residing in each compartment of said library. Therefore in a specific embodiment, the libraries may be used in a method for determining the function of a naturally occurring polynucleotide sequence, said method comprising: a) transfecting a host cell with a vector according to the invention wherein the vector encodes an RNA molecule including a first stretch of 19 consecutive nucleotides homologous to a portion of the naturally occurring polynucleotide, and b) detecting a change in cellular phenotype.

Each vector in the library may be introduced into one or more cells and changes in protein expression, or phenotype observed. Methods are described for infecting a host with the adenoviral vectors that express the RNA molecules including the stretch of 19 consecutive nucleotides in the host, identifying an altered phenotype induced in the host by the knockdown of the expressed RNA molecules, and thereby assigning a function to the product(s) encoded by the expressed RNA molecules. The methods can be fully automated and performed in a multi-well format to allow for convenient high throughput analysis of expressed RNA molecules.

As used herein, the term "homologous" refers to a nucleic acid which has a nucleotide sequence wherein 80%, preferably 85%, preferably 90% or more preferably 95% of nucleotides are identical to a stretch of consecutive nucleotides present in the RNA-molecule to be silenced. For example, in a sequence with 95% homology the nucleotide sequence is identical to the reference nucleotide sequence, i.e. the RNA molecule, except that the nucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence of at least 95% homology to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

Specifically, as used herein the with reference to nucleotides as disclosed herein, it is intended that these are at least 80% identical to the corresponding nucleotides in a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced, or alternatively that preferably 85%, preferably 90% or more preferably 95% of nucleotides disclosed herein are identical to the corresponding nucleotides in a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced. In a specific embodiment, the present invention provides a polynucleotide in which all 19 nucleotides in said first stretch of 19 consecutive nucleotides are identical to a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced.

In a suitable manner of the present invention the first and second stretch of 19 consecutive nucleotides may form a dsRNA molecule.

The term identical refers to two stretches of nucleotides that have at each position of the stretch the same nucleotide, meaning that if one stretch has an A-nucleotide at position three, the other stretch of nucleotides also has an A-nucleotide at position three. This is applicable for each nucleotide in both stretches.

The term polynucleotide refers to a nucleic acid sequence. A polynucleotide can be a DNA-or RNA-nucleic acid sequence. It may have natural occurring nucleotides but also chemically modified nucleotides.

Brief Description of the Drawings

Figure 1 is a schematic representation of the cloning strategy for vector construction showing utilization of SapI sites and an E colldeath gene.

Figure 2 is a schematic representation of the vector construction for cloning specifically it shows adenoviral vector development for 56 nt inserts. The schematic presentation of the oligos used for the vector construction is also given.

Figure 3 is a schematic presentation of the knock-down vector pKD122.

The various aspects of the present invention are further illustrated in the following non-limiting

Examples.

Example 1: selecting target sequences: The polynucleotides of the present invention are built from the stretches of 19 consecutive nucleotides as explained in Example 2. The design for the 19 consecutive nucleotide sequences that form part of the polynucleotides of the present invention is fully automated. A program has been developed that looks for candidate sequences of 19 consecutive nucleotides. These 19 nucleotide sequences are searched in a target cDNA sequence. The program searches 19 nucleotide sequences according to the following scheme: 1. The search for the 19 nucleotide sequences starts 75 nucleotides downstream from the start codon of the cDNA. If a start codon is not known or available, the search starts 75 nt downstream of the first ATG. If no ATG is present, the 19 nucleotide sequences are searched starting from the first nucleotide of the cDNA sequence.

2. The cDNA sequence is scanned from 5' to 3' direction and when a 19 nucleotide sequence is found that conforms to an (N18)C pattern, it is retained (N=any nucleotide). This procedure is stopped 19 nucleotides before the end of the cDNA sequence.

3. Then, the candidate 19 nucleotide sequence is checked for the presence of 4 consecutive identical nucleotides. If this pattern is found, this candidate 19 nucleotide sequence is rejected and the program starts at step 2 again.

4. At this point, the 19 nucleotide sequences that are found are attributed a score of 0.

5. If the 19 nucleotide sequence is not rejected in step 3, the percentage of G and C nucleotides of the candidate 19 nucleotide sequence is determined. The percentage of G and C nucleotides should be between 30 and 70%. If it is not between 30 en 70%, the 19 nucleotide sequence is rejected and the procedure starts at step 2 again.

6. The 19 nucleotide sequence is checked for a Pac site (1TAATTAA) and will be rejected if this pattern is found, starting again from step 2.

7. If the 19 nucleotide sequence contains an unspecified nucleic acid, the sequence will be rejected and the design will start from step 2 again.

8. The candidate 19 nucleotide sequence is checked for the presence of 3 consecutive A-or T-nucleotides. If this pattern is found, the score of the 19 nucleotide sequence is decreased by 5 units.

9. The 19 nucleotide sequence is then searched against a number of public sequence databases. The blast algorithm, (the National Center for Biotechnology Information (NCBI)) is used to match the 19 nucleotide sequence against; a. -a human cDNA database, consisting of the complete RefSeq collection. For every extra cDNA sequence, apart from the target cDNA sequence or any cDNA derived from the same locus of the target cDNA, that fully matches the 19 nucleotide sequence, the score of the 19 nucleotide sequence is decreased with the 300 units. For every cDNA sequence, that has 1 or 2 mismatches compared to the target, the score of the 19 nucleotide sequence is decreased with (3 -(number mismatches, 1 or 2)) * 10. This formula takes into account that 1 mismatch is more likely to give an unwanted off target effect (and therefore it gets a higher penalty score that is subtracted from the total score).

b. -the mouse RefSeq database or -the rat RefSeq database The score of the 19 nucleotide sequence is increased with 5 units for every matching mouse or rat cDNA sequence.

10. If the number of A's or T's in the last 5 positions of the 19 nucleotide sequence is higher than 3, an additional 16 units will be added to the final score.

11. If position 5 of the 19 nucleotide sequence is an A, an additional 3 units will be added to the final score.

12. If position 12 of the 19 nucleotide sequence is a T, an additional 3 units will be added to the final score.

13. If position 15 of the 19 nucleotide sequence is a G, an additional 3 units will be added to the final score.

14. If the target for the 19 nucleotide sequence has multiple transcript variants, an additional score is calculated as: number of transcript variants targeted with the nucleotide sequence/total number of transcript variants * 10.

15. Procedure 2 until 14 is repeated until the end of the cDNA is reached.

16. Finally, an output report is generated. In this report, the cDNA sequence is graphically represented, together with all the designed 19 nucleotides sequences. In a second part of the report, the designed 19 nucleotide sequences are listed with the characteristics mentioned in point 1 to 14. These include the matching human, mouse and rat transcript sequences and the outcome of each of the pairwise comparisons to the target cDNA sequence.

The program performing the rules described above has been written in perl (practical extracting and reporting language). In our case extensive use has been made of the bioperl' bioinformatics toolkit (Pen, O'Reily.com). However, a person skilled in the art can use another language to implement the rules set above in a program and use this program to design sIRNA that give an effective knock-down effect.

Example 2: S1RNA expression constructs: 2.1 Non viral 5IRNA express/on constructs: The construction of the 5IRNA expression constructs is depicted in Figure 1. In short, oligos containing knock-down target sequences as depicted in Table 1 (19 nt) are cloned in the knock-downvector, pKD122. This non-viral DNA expression plasmid can be introduced using DNA transfer methods known in the art, such as lipofectamine or PEI. The individual knockdown constructs for each gene can be pooled or can be used separately.

2.2 adeno viral 51RNA express/on constructs The 5iRNA expression constructs can also be contained in viruses. The viruses can be made in an arrayed format, if preferred. The arrayed viruses mediate expression of the 5iRNA constructs; each welt contains a unique recombinant virus carrying a 5iRNA expression construct targeted against a gene, i.e. one target gene per well. Further details about the concept of arrayed adenovirat vectors can be found in WO 99/64582, US 6,340,595 and US 6,413,776 (Arrayed adenovira I libraries for performing functional genomics).

In addition to the knock-down vector, pKD122, two other materials are needed for the generation of recombinant adenovirus particles: a helper cosmid and a packaging cell line (see also WO 99/64582 and us 6,340,595). The cosmid (pWE/Ad.AflII-rITRAE2A) contains the main part of the adenovirus serotype 5 genome (bp 3534-35953) from which the E2A gene is deleted.

The Per.C6/E2A packaging cell line (Crucell NV) is derived from human embryonic retina cells (HER) transfected with plasmids mediating the expression of the El and E2A genes. The adenoviral genes that are integrated into the genome of the PER.C6/E2A cell line share no homology with the adenoviral sequences on the knock-down plasmid and the cosmid.

Consequently, vector stocks that are free of replication competent adenoviruses (RCA5) are prepared.

To obtain viruses, the knockdown plasmid is co-transfected with the helper cosmid into a packaging cell line PER.C6/E2A. Once these plasmids are transfected into the PER.C6/E2A cell line, the complete Ad5 genome (except the El and E2A genes) is reconstituted by homologous recombination. The helper and knock-down plasmids contain homologous sequences (bp 3535- 6093), which are a substrate for this recombination event.

2.3 Design of oligos: Oligonucleotides are designed to specifically target mRNAs of interest. The selected target sequences are listed in Table 1 and are used for the construction of knock-down adenoviral expression clones. Specific pairs of forward (F) and reverse (R) oligonucleotides are annealed together to form a duplexed structure that is necessary for cloning into the knock-down vector (see figure 2). The 56 nt oligos containing knock-down target sequences have the following structure the Forward oligonucleotide: 5'-ACC-Nl9* GTTTGCTATAAC (SEQ ID NO: 7472) -N19--TTT-3' Reverse ol igonucleotide: 5'TAAAAANl9*GTTATAGCAAAC (SEQ ID NO: 7473)-N19-3' Where N19 represents the DNA sequences corresponding to the sequences as depicted in Table 1, and N19* is the reverse-complement sequence of this sequence.

The single stranded oligonucleotide components are synthesized and delivered by standard manufacturers of molecular biology products (e.g. Sigma or Invitrogen) in a desalted and mixed form at a final concentration of 50 pmol/pL of each corresponding forward and reverse oligonucleotide in 96 or 384 well plates. In order to generate the double stranded oligonucleotides 2 pL annealing buffer (NEBuffer 2, lOx concentrated, Biolabs) is added to 18 pL oligos in a 96 well PCR plate. The plates are spun down briefly and subsequently sealed. The plates are incubated in the PCR machine for 5' at 95 C and slowly cooled to room temperature.

The annealed oligos are diluted l000x in ddH2O before ligation into the knock-down expression vector.

2.4 Design of pKDJ22 The knock-down expression vector, pKD122 (Figure 3) is based on the plPspAdapt6 (WO 99/64582). The plPspAdApt6 plasmids contain the 5' part (bp 1-454 and bp 3511-6093) of the adenovirus serotype 5 genome from which the El gene is deleted and a promoter is introduced.

In contrast, to the plasmid plPspAdApt6, the siRNA expression vectors of plPspAdapt lack the CMV promoter, the SV4O polyadenylation site and the larger part of the polylinker. pKD122 further contains a U6 promoter, Sap I endonuclease recognition sites and the E.Coil lethal gene, ccdB.

The endonuclease Sap I is capable of digesting dsDNA adjacent to its recognition sites (GCTCTTC(N)114) and creates a linearized plasmid of which the 3' overhangs are not compatible (see Figure 2)and 2 Sap I sites flanking the ccdB gene fragment. This has the advantage that the DNA ends of the knock down construct cannot be ligated to itself and that the Sap I sites themselves are completely removed from the final construct. For these reasons SapI is used for the construction of expression plasmids. The ccdB, is included in the fragment such that when the restriction fragment is not correctly excised or not completely purified from the linearized vector the ccdB gene remaining in the plasmid, after transformation, no E. coil colonies are formed. Only E. coil containing correct expression plasmids with the two unique SapI overhangs and without the ccdB gene will form colonies.

plPspAdapt6 is grown in the methylase negative E. coil strain DM1 to prevent methylation of the XbaI-site located at the 3'end of the polyAdenylation site. The DNA is isolated and digested with Xba I and religated to remove the 142 bp fragment containing the polyAdenylation signal. The religated vector is called plPspAdapt6-deltaPolyA. The polylinker is removed from plPspAdapt6-deltaPolyA by digestion with EcoRI and BamHI, blunted with Klenow and relegated. This religated vector is called plPspAdapt6-deltaPolyA delta-polylinker.

plPspAdapt6-deltaPolyA-delta polylinker is digested with AvrH and HindlIl to remove the CMV and the 676 bp ccdB gene fragment was isolated from plPspAdaptlOZeoDestA (WO 99/64582) with BamHI and Sal I. The genomic human U6 gene (Accession number M14486 (GenBank, NCBI)) is cloned by a PCR based strategy using human genomic DNA. The region cloned starts at nucleotide -265 5' of the transcription start site until nucleotide +198 3'of the transcription start site. The primers used are: 5'-GcacgTTCTAGAAGGTCGGGCAGGAAGAGGGCCT-3' (SEQ ID NO: 7474) 5'-ccgtgcMGC I I I GGTAAACCGTGCACCGGCGTA-3' (SEQ ID NO: 7475) The PCR product is cloned into the Xba I and Hind III sites of plPspAdapt6-deltaPolyA-delta polylinker, the resulting vector is hU6(+1)plPspAdapt6-dpA delta polylinker.

Two U6 Sap I PCR fragments (a left, L, and a right R) containing the U6 promoter sequences together with the SapI recognition sequences are made with the following primers: 5'-CGACCATGCGCGGATCCGCTCTTCTGGTGTTTCGTCCTT-3' (SEQ ID NO: 7476) 5'-CGGATCCGCGCATGGTCGACGCTCTTCATTACATCAGGTTGTrT-3' (SEQ ID NO: 7477).

(SEQ ID NO: 7474) with (SEQ ID NO: 7476) gives the L fragment and (SEQ ID NO: 7475) with (SEQ ID NO: 7477) gives the R fragment with the hU6(+1) plPspAdapt6-dpA delta polylinker as template.

The R-fragment is digested with XbaI and BamHl, and the L-fragment is digested with SalT and Hindill. plPspAdapt6-deltaPolyA- delta polylinker was digested with AvrII and Hindlil. The digested R-and L-fragments together with the digested hU6(+ 1) plPspAdapt6-deltaPolyA delta polylinker and the ccdB fragment are ligated with 14 DNA ligase in ligase buffer (about 30 ng of each fragment in the ligation) and transformed in DB3.1 cells (wherein the ccdB is not toxic, Invitrogen) A colony PCR is performed to check sequences with primers SEQ ID NO: 7472 and 7473. This should generate a 1000 bp fragment. Positive clones are digested with Hincli and BglII, the correct clones give fragments of 3800, 1400, 538, 402 and 134 bp in size. Clones that give these fragments were confirmed by sequence analysis. The resulting vector is pKD122 (Figure 3).

2.5 aon/ng of the oligos The knockdown vector pKD122 (Figure 3) is digested by Sap I and gel purified. Digestion mix: pL restriction buffer Neb 4, 10 pL Sap I in 300 pL total volume for 9 pg of knock-down vector, and incubated at 37 C over night. Gel: 1 % agarose in lx TAE, 2 pL lOx loading buffer to 5 pL of digestion mix; the digested vector is isolated from gel with QlAquick gel extraction kit (Qiagen).

Ligation of the annealed oligos in the knock-down vector: 0.5 pL digested knock-down vector (40 ng/pL), 1 pL 14 DNA ligase buffer (lOx concentrated, New England Biolabs) 0.5 pL T4 DNA ligase (New England Biolabs) and 7 pL milliQ H20 are added per well. Added to this is 1 pL of the diluted annealed oligos. The plates are incubated over night at room temperature.

2.6 Transformation: 1-2 pL of each ligation mix is put into a new 96-well PCR plate and put on ice. 25 pL competent DH5a-cells (Subcloning efficiency, Invitrogen) are added and incubated on ice for 30 minutes.

The bacteria are heat shocked for 40 seconds at 37 C and put on ice for 2 minutes. The bacteria are allowed to recover by adding 170 pL SOC-medium (room temperature; Invitrogen) to each well and incubation for 1 hour at 37 C in a rotary shaker at 100-150 rpm. Cells are spun down at 1700 g for 1 mm. 100 pL supernatant is taken to be discarded and the bacteria in the pellet are resuspended in the remainder of the supernatant (100 pL). 50 pL of the cell suspension (50%) is plated in 1 well of a 6-wells plate (filled with 3 mL LB agar+100 j.ig/mL ampicillin /well). The plates are incubated overnight at 37 C.

2.7 Colony picking: 3 colonies of each knock-down vector construct are picked and inoculated as both agar-stab (LB agar with 100 pg/mL ampicillin) and liquid culture (LB medium with 100 pg/mL ampicillin). The clones in the agar-stab are used for confirmation of their sequence by sequence analysis. The DNA isolated from the Liquid cultures of those clones with the correct sequence is isolated and transfered to a new 96 well plate to be linearized by digestion with the endonuclease PI-PspI.

PI-PspI digestion mix (lx) contains 0.5 pg annealed knock-down vector 2.5 pL PI-PspI enzyme, (1U/ pL Biolabs), 2.5 pL PI-PspI NEBuffer, (lOx concentrated Biolabs), 0.25 pL BSA (lOOx concentrated Biolabs) in a total volume of 25 pL (end concentration = 20 ng/pL). The mixture is incubated over night at 65 C in a humified box. The digestion is checked on gel: 10 pL PI-PspI digestion mix, added to this is 2 pL loading buffer (lOx concentrated), and put on gel (l% agarose in lx TAE buffer + ethidium bromide).

2.8 Transfection Each clone is co-transfected to PER.C6/E2A cells together with the cosmid pWE/Ad.AflhI-rITRAE2A (W09964582. US 6,340,595. CPE is scored 14 days after transfection. After the final scoring the plates are stored at -80 C until further propagation of the viruses.

2.9 Virus propagation The final virus propagation step is aimed at obtaining a higher percentage of wells showing cytopatic effect and more homogenous virus titers. Viruses are propagated according to the following procedure. The transfection plates stored at -80 C are thawed at room temperature for about 1 hour. By means of a 96 channel Hydra dispenser (Robbins), 20 pL of the supernatant is transferred onto PER.C6/E2A cells seeded in 96 well plates at a density of 2.25x104 cells/well in 180 tl of DMEM supplemented to contain lO% FBS and 2 mM MgCl2. Cells are incubated at 34 C, 10% CO2 for approximately 10 days and the number of wells showing cytopatic effect is scored. In general, the number of wells showing cytopatic effect is increased after propagation. The plates are then stored at -80 C. (

In addition, modifications to the viral coat proteins can be introduced to obtain a different or improved tropism (EP 1191105).

The individual knockdown adenoviruses can be used as arrays but also can be pooled to various degrees i.e. sets of pools or one large pool.

Claims

Claims 1. Polynucleotide comprising an RNA sequence comprising a first

stretch of 19 consecutive nucleotides and a second stretch of 19 consecutive nucleotides, which is the reverse-complement of the first stretch of 19 consecutive nucleotides, wherein in said second stretch of 19 consecutive nucleotides: a. in the 51 -> 3' direction, the last nucleotide is a C-nucleotide; and b. no stretches of four or more consecutive identical nucleotides are present; c. the total number of G-and C-nucleotides is between 3O7O% of the total number of nucleotides d. in the 5' -> 3' direction, consecutive nucleotides 1-19 are homologous to a RNA-molecule.
2. A polynucleotide according to claim 1, wherein the RNA-molecule is a human RNA molecule.
3. A polynucleotide according to claim 1 or 2, wherein in the second stretch of 19 consecutive nucleotides no stretches of three or more consecutive A-nucleotides are present
4. A polynucleotide according to claim 1 or 2 wherein in the second stretch of 19 consecutive nucleotides no stretches of three or more consecutive U-nucleotides are present
5. A polynucleotide according to any one of claims 1-4, wherein, in the 5' -> 3' direction, consecutive nucleotides 1-19 of the second stretch of 19 consecutive nucleotides are unique.
6. A polynucleotide according to any one of claims 1-5, wherein, in the 5' -> 3' direction, consecutive nucleotides 1-19 of the second stretch of 19 consecutive nucleotides are homologous to a sequence positioned at least 75 nucleotides downstream of the translation initiation site of the transcribed RNA molecule encoding a polypeptide.
7. A polynucleotide according to any one of claims 1-6, wherein, in the 5' -> 3' direction, consecutive nucleotides 1-19 of the second stretch of 19 consecutive nucleotides are homologous to a sequence positioned at least 50 nucleotides upstream of the translation termination site of the transcribed RNA molecule encoding a polypeptide.
8. A polynucleotide according to any one of claims 1-7 wherein the RNA sequence also comprises a linker sequence linking the first stretch of 19 consecutive nucleotides with the second stretch of 19 consecutive nucleotides.
9. A polynucleotide according to claim 8, wherein the linker sequence is 4-30 nucleotides long, preferably 5-15 nucleotides long and most preferably 12 nucleotides long
10. A polynucleotide according to claim 8 or 9, wherein the linker sequence is SEQ ID NO:
11. A polynucleotide according to any one of claims 1-10, wherein the second stretch of 19 consecutive nucleotides is selected from a group consisting of SEQ ID NO: 1-7470.
12. A vector capable of transfecting a host cell and comprising a sequence encoding a polynucleotide according to any one of claims 1-11 and a promoter sequence operatively linked to the sequence encoding the polynucleotide.
13. A vector according to claim 12, wherein the promoter is a microRNA promoter, preferably a let-7 promoter.
14. A vector according to claim 13, wherein the promoter is a promoter recognized by RNA Polymerase III, preferably U6 small nuclear RNA.
15. A vector according to any one of claims 12-14, wherein the vector is an adenoviral vector, preferably the adenoviral vector is replication defective.
16. A library of polynucleotide sequences according to any one of claims 1-11.
17. A ibrary of vectors according to any one of claims 12-15.
18. A library according to claim 17 wherein the vectors are viral vectors preferably selected from a group consisting of MV, Lentivirus or Retrovirus.
19. A library according to claim 18 wherein the vectors are adenoviral vectors, preferably the adenoviral vectors are replication defective.
20. A method of making a vector according to claim 15 comprising the steps: a. synthesizing a forward primer with the following sequence in the 5' -> 3' direction: the nucleotides ACC ii. the reverse-complement sequence corresponding to the DNA sequence from step a-iv iii. the nucleotides GT1TGCTATAAC (SEQ ID NO: 7272) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 v. the nucleotides lTr b. synthesizing a reverse primer with the following sequence in the 5' -> 3' direction the nucleotides TAAAAA ii. the reverse-complement sequence corresponding to the DNA sequence from step b-iv iii. the nucleotides GTTATAGCAAAC (SEQ ID NO: 7273) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 c. annealing the primers from step (a) and (b) 1 0 d. exchanging the ccdB sequences in plasmid pKD122 for the annealed primers from step c.
21. Method of preparing a vector according to claim 14 comprising the steps: a. synthesizing a forward primer with the following sequence in the 5' -> 3' direction: i. the nucleotides ACC ii. the reverse-complement sequence corresponding to the DNA sequence from step a-iv iii. the nucleotides GT1TGCTATAAC (SEQ ID NO: 7272) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 v. the nucleotides liT b. synthesizing a reverse primer with the following sequence in the 5' -> 3' direction the nucleotides TAAAAA ii. the reverse-complement sequence corresponding to the DNA sequence from step b-iv iii. the nucleotides G1TATAGCAAAC (SEQ ID NO: 7273) iv. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 1-7470 c. annealing the primers from step a. and b., d. exchanging the ccdB sequences in plasmid pKD122 for the annealed primers from step c.,
22. A method of determining the function of a naturally occurring polynucleotide sequence comprising transfecting a host cell with a vector according to any one of claims 12-15, the vector transcribing a polynucleotide sequence according to any one of claims 1- 11 and detecting a change in cellular phenotype.
23. Method of determining the function of a naturally occurring polynucleotide sequence in a high throughput setting, a. providing a library of vectors according to any one of claims 17-19 b. transducing a host cell with the vectors of step (a), c. expressing in the host cell the product(s) of the vectors of step (a), d. thereby altering a phenotype of the host, e. identifying the altered phenotype and, f. assigning a function to the naturally occurring polynucleotide sequence (s).