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WO2004094636A1 - Constructions demontables effectives d'arnsi - Google Patents

Constructions demontables effectives d'arnsi

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Publication number
WO2004094636A1
WO2004094636A1 PCT/EP2003/004362 EP0304362W WO2004094636A1 WO 2004094636 A1 WO2004094636 A1 WO 2004094636A1 EP 0304362 W EP0304362 W EP 0304362W WO 2004094636 A1 WO2004094636 A1 WO 2004094636A1
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WIPO (PCT)
Prior art keywords
nucleotides
sequence
stretch
nmj
polynucleotide
Prior art date
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Ceased
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PCT/EP2003/004362
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English (en)
Inventor
Gerardus Johannes Franciscus Arts
Jan Van Der Schueren
Mark Jacques Yvonne Lambrecht
Kristina Djokic
Remko Johannes Clasen
Emir Mesic
Sandra Griffioen
Carolina Johanna Leonarda Bergs
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Galapagos Genomics NV
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Galapagos Genomics NV
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Priority to PCT/EP2003/004362 priority Critical patent/WO2004094636A1/fr
Priority to AU2003224132A priority patent/AU2003224132A1/en
Publication of WO2004094636A1 publication Critical patent/WO2004094636A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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    • C12N2310/53—Physical structure partially self-complementary or closed
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    • C12N2330/31—Libraries, arrays

Definitions

  • 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 interfering polyribonucleic acid molecules .
  • RNA interference is the post-transcriptional process of gene silencing mediated by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA and is observed in animals and plants.
  • dsRNA double stranded RNA
  • 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.
  • siRNAs small interfering RNAs
  • RNAi 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 (>30 base pairs) dsRNA 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.
  • Long dsRNA is also known to activate the interferon-induced (2' -5') oligoadenylate synthetase. Upon activation, this enzyme polymerizes ATP into 2' -5' -linked nucleotide oligomers (also indicated by 2-5A) .
  • 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.
  • siRNA transfection approach An important bottleneck in the siRNA transfection approach is its limited applicability to target different cell types, especially primary cells.
  • Primary cells are closest to the in vivo situation and often have the highest physiological relevance.
  • Non-viral DNA or siRNA transfection technologies have severe limitations with regard to these cells and are not efficient and reliable. Practical use of these approaches needs significant optimisation of conditions, and in general lack the robustness necessary for large-scale applications.
  • the gene transfer reagents used are often toxic, yielding lower levels of viable transduced cells. In essence, they do not allow a generic siRNA 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.
  • transfection of the siRNA gives a short knockdown effect. For a prolonged knock-down effect in cells several additional transfections are necessary.
  • WO03020391 describes adenoviral vectors which express hairpin RNAs 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.
  • 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. Some RNAi ' s have a 10-fold effect, some have seen a 50-fold effect, and some don't work at all. For example , a siRNA directed against vi entin (nt 346-368 from Genbank (NCBI) : NM_003380 relative to start codon) did not give a silencing effect.
  • 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 al 2001)) .
  • a procedure for designing siRNAs for efficiently inducing RNAi in mammalian cells has been suggested (Elbashir et al 2002) .
  • a siRNA against c-myc, designed according to this protocol was uneffective 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 RNAi by siRNAs.
  • the present invention provides polynucleotides that are very effective in the silencing a RNA molecule.
  • the present invention provides polynucleotide comprising an RNA sequence comprising a first stretch of 21 consecutive nucleotides and a second stretch of 21 consecutive nucleotides, complementary to the first stretch of 21 consecutive nucleotides, wherein in first stretch of 21 consecutive nucleotides: a. in the 5' -> 3 ' direction, the first nucleotide is an A-nucleotide , the second nucleotide is a C-nucleotide and 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 33-71% of the total number of nucleotides; d. in the 5' -> 3 ' direction, consecutive nucleotides 3- 21 are homologous to a RNA-molecule.
  • the total number of G- and C-nucleotides can be 33, 35, 40, 45, 50, 55, 60, 65, 70, and 71 % of the total number of nucleotides.
  • the consecutive nucleotides 3-21 in the 5' -> 3' direction of the first stretch of 21 consecutive nucleotides are homologous to the RNA-molecule to be silenced.
  • “Homologous” to the RNA-molecule can mean that nucleotides 3-21 of the stretch of 21 consecutive nucleotide are identical to a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced, or that 95%, 90%, 85% or 80% of the nucleotides 3-21 of the stretch of 21 consecutive nucleotide are identical to a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced.
  • a polynucleotide with all 21 nucleotides of the stretch of 21 consecutive nucleotide identical to a stretch of 21 consecutive nucleotides present in the RNA-molecule to be silenced is also possible.
  • the first and second stretch of 21 consecutive nucleotides form a double stranded RNA molecule.
  • the invention provides polynucleotides wherein the RNA-molecule is a human RNA molecule.
  • the RNA molecule will be silenced by the polynucleotides of the present invention.
  • the RNA molecule to be silenced can be a drugable gene.
  • Drugable genes are genes with a pharmaceutical value, ie that can be used to discover and develop small molecule drugs. New drugable genes can be found on the bases on their similarity to proteins which have proven amenable to small molecule compound development in the past.
  • Known drugable genes include but ar not limited to: G- protein coupled receptors (GPCRs), ion channels, nuclear hormone receptors, kinases, phosphatases, proteases and other enzymes .
  • the invention provides polynucleotides wherein in the first stretch of 21 consecutive nucleotides no stretches of three or more consecutive A- nucleotides are present.
  • the invention provides polynucleotides wherein in the first stretch of 21 consecutive nucleotides no stretches of three or more consecutive U-nucleotides are present
  • the invention provides polynucleotides, wherein, in the 5' -> 3' direction, consecutive nucleotides 3-21 of the first stretch of 21 consecutive nucleotides are unique. These unique oligonucleotides are homologous to a stretch of 21 consecutive nucleotides that is found only once in known sequences. Sequence databases can be searched to check if these sequences occur only once. Known databases include the EST database, the EMBL nucleotide sequence database, GenBank, and the Entrez nucleotide database, but there are many more sequence databases and all of these can be used.
  • the polynucleotide with the unique consecutive nucleotides 3-21 in the 5' -> 3' direction, of the first stretch of 21 consecutive nucleotides will silence only one specific mRNA molecule.
  • the consecutive nucleotides 3-21 in the 5' -> 3' direction, of the first stretch of 21 consecutive nucleotides are found more than once in sequence databases.
  • These polynucleotides can silence alternative gene transcripts of the same sequence or alternative splicing variants.
  • the stretch of 21 consecutive nucleotides can be designed in such a way that the sequence is found in more than one member of a family of proteins.
  • the polynucleotides containing this stretch of 21 consecutive nucleotides can silence more than one member of a family of proteins .
  • polynucleotides wherein, in the 5' -> 3' direction, consecutive nucleotides 3-21 of the first stretch of 21 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.
  • polynucleotides are provided wherein, in the 5' -> 3' direction, consecutive nucleotides 3-21 the first stretch of 21 consecutive nucleotides homologous are to a sequence positioned at least upstream of the translation termination site of the transcribed RNA molecule encoding a polypeptide.
  • RNA sequence also comprises a linker sequence linking the first stretch of 21 consecutive nucleotides with the second stretch of 21 consecutive nucleotides.
  • the linker sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 8 nucleotides long.
  • linker sequence is UUGCUAUA (SEQ ID NO: 1) .
  • the invention provides polynucleotides, wherein the first stretch of 21 consecutive nucleotides is selected from a group consisting of SEQ ID NO: 2-11888.
  • the invention provides polynucleotides, wherein the first stretch of 21 consecutive nucleotides is selected from a group consisting of SEQ ID NO: 1342, 1338, 1343, 633, 635.
  • Another embodiment of the invention provides vector capable of transfecting a host cell and comprising a sequence encoding the polynucleotides of the present invention and a promoter sequence operatively linked to the sequence encoding the polynucleotide.
  • the promoter is a microRNA promoter, more preferably a let-7 promoter.
  • the promoter is a promoter recognized by RNA Polymerase III, more preferably U6 small nuclear RNA.
  • a person skilled in the art can use other promoters recognized by Polymerase III for the vectors of the present invention such as, HI, tRNA, snRNA, VA RNA, 5S rRNA.
  • vectors are provided, wherein the vector is an adenoviral vector, preferably the adenoviral vector is replication defective.
  • the replication defective adenoviral vectors are El-deleted, and/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, MoMuLV ("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 AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells.
  • 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 polynucleotide of the present invention.
  • the library of the present invention provides vectors according to the present invention.
  • the vectors may comprise plasmids, naked RNA.
  • the vectors are viral vectors preferably selected from a group consisting of AAV, Lentivirus or Retrovirus.
  • more than one vector thereby introducing more than one stretch of 21 consecutive nucleotides can be introduced into a single host cell.
  • 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 single element, compartmentalized, or discrete elements. Alternatively, a library comprising pools of vectors may be prepared. Methods are described for making a vector comprising the synthesis a forward primer and a reverse primer.
  • the forward primer is synthesized with the following sequence: in the 5' -> 3 ' direction: i. the nucleotides ACC ii. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 2-11888 iii. the nucleotides TTGCTATA iv. the antisense sequence corresponding to the DNA sequence from step a-ii v.
  • the reverse primer is synthesized 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: 2-11888 iii. the nucleotides TATAGCAA iv. the antisense sequence corresponding to the DNA sequence from step b-ii 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: 2-11888 can be reversed.
  • the forward and reverse primers look then as follows : a. forward primer with the following sequence in the 5' -> 3' direction: i. the nucleotides ACC ii. a DNA sequence corresponding to a sequence selected from the group consisting of SEQ ID NO: 2-11888 iii. the nucleotides TTGCTATA iv. the antisense sequence corresponding to the DNA sequence from step a-ii v. the nucleotides TTT b. a reverse primer with the following sequence in the 5' -> 3' direction i. the nucleotides TAAAAA ii.
  • the invention further provides methods of determining the function of a naturally occurring polynucleotide sequence comprising transfecting a host cell with a vector according to claims 12-15, the vector transcribing a polynucleotide sequence according to claims 1-11 and detecting a change in cellular phenotype. According to a preferred embodiment of the present inventions methods of determining the function of a naturally occurring polynucleotide sequence in a high throughput setting are provided wherein, a.
  • 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.
  • determining the function of a naturally occurring polynucleotide sequence comprising transfecting a host cell with a vector according to the invention, the vector, encoding a RNA molecule including a stretch of 21 consecutive nucleotides homologous to a portion of the naturally occurring polynucleotide and 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 21 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 multiwell format to allow for convenient high throughput analysis of expressed RNA molecules .
  • RNA molecule refers to a nucleic acid having a nucleotide sequence of which 95%, 90%, 85% or 80% of the nucleotides 3-21 of the stretch of 21 consecutive nucleotide are identical to a stretch of 19 consecutive nucleotides present in the RNA-molecule to be silenced.
  • nucleotide sequence is identical to reference nucleotide, i.e. the RNA molecule, except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • 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 .
  • 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 a A- nucleotide on position three, the other stretch of nucleotides also has a A-nucleotide on position three. This is applicable for each nucleotide in both stretches.
  • polynucleotide refers to a nucleic acid sequence.
  • a polynucleotide can be a DNA-, RNA-, peptide nucleic acid sequence. It may have natural occurring nucleotides but also chemically modified nucleotides.
  • Figure 1 is a schematic representation of the cloning strategy for vector construction showing utilization of Sapl sites and an E. coli death gene.
  • Figure 2 is a schematic representation of the vector construction for cloning. 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.
  • Figure 4 is a graph showing the functional knock-down of GNAS .
  • Adenoviral constructs encoding hairpin siRNA targeted against GNAS give a specific knock-down of GNAS on the functional level.
  • Figure 5 is a graph showing the functional knock-down of SYK as measured by the released beta-hexosaminidase .
  • FIG. 6 shows a graph demonstrating that adenoviral mediated knock-down of IKK ⁇ leads to reduced levels of phosphoryated I ⁇ B ⁇ , an I ⁇ B kinase substrate, upon TNF ⁇ induction.
  • Western detection of IKK ⁇ and ⁇ -tubulin in the samples derived from U20S cells (no virus infection, lanes 1- 3; infected with adenovirus Ad-IKK- ⁇ , lanes 4-6) harvested at different time points after 50 ng/ml TNF ⁇ stimulation (0 minutes, lanes 1, 4, or 10 minutes, lanes 2, 5, or 30 minutes, lanes 3, 6) .
  • Detection of phosphorylated I ⁇ B ⁇ by Bio-Plex phosphoprotein analysis of the same samples as described.
  • the phosphorylation state of I ⁇ B ⁇ is given as Mean Fluorescent Intensity (M.F.I.) at the vertical axis.
  • M.F.I. Mean Fluorescent Intensity
  • Figure 7 shows a graph demonstrating the functional knock-down of MMP2.
  • the effect of adenoviral knock-down was determined by RT-PCR and at the level of protein activity. Gelatin-degrading activity of MMP2 was measured using 10% gelatin-zymogram gels.
  • Ad-MMP2 an adenoviral cDNA expression construct encoding the MMP2 protein
  • Example 1 selecting target sequences:
  • the polynucleotides of the present invention are built from the stretches of 21 consecutive nucleotides as explained in example 2.
  • the design for the 21 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 21 consecutive nucleotides. These 21 nucleotide sequences are searched in a target cDNA sequence.
  • the program searches 21 nucleotide sequences according to the following scheme:
  • the candidate 21 nucleotide sequence is checked for the presence of 4 consecutive identical nucleotides. If this pattern is found, this candidate 21 nucleotide sequence is rejected and the program starts at step 2 again.
  • the candidate 21 nucleotide sequence is checked for the presence of 3 consecutive A- or T-nucleotides . If this pattern is found, the score of the 21 nucleotide sequence is decreased with 5 units. 7.
  • the 21 nucleotide sequence is then searched against a number of public sequence databases.
  • the blast algoritm (the National Center for Biotechnology Information (NCBI) ) is used to match the 21 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, that matches the 21 nucleotide sequence , the score of the 21 nucleotide sequence is decreased. The score is decreased with 10 units for the first additional matching cDNA sequence and with another 4 units for every additional matching cDNA sequence.
  • b. -the mouse RefSeq database c. -the rat RefSeq database The score of the 21 nucleotide sequence is increased with 35 units for every matching mouse or rat cDNA sequence.
  • the 21 nucleotide sequence is then searched against the human ⁇ Expressed Sequence Tag' (EST) database. If the 21 nucleotide sequence does not match any ESTs, it score is decreased by 10 units. If the 21 nucleotide sequence matches perfectly to an EST, the complete EST is matched against the target cDNA sequence.
  • the program used for this pairwise sequence comparison is blast2seq, available from NCBI. From this pairwise sequence comparison, one can determine the sequence similarity of the EST and cDNA (p- value) . If the p-value is e ⁇ 100 or lower than the EST is similar to the cDNA target sequence, if the p-value is more than e ⁇ 100 than the EST is dissimilar to the cDNA sequence.
  • the 21 nucleotide sequence does match ESTs, its score is decreased by 50 units for the first dissimilar EST and another 5 units for every extra dissimilar EST. The score of the 21 nucleotide sequence is increased with one unit for every EST sequence similar to the target cDNA sequence.
  • Each 21 nucleotide sequence has a score. All possible combinations of 4 of the designed 21 nucleotide sequence (so called sets' ) are made. For each set a combined score is calculated according to the following formula : F (oligo 1-n) - T + ( ( ⁇ x * ..
  • the 3 highest scoring 21 nucleotide sequences are used to design the oligos for the plasmid construction as described in example 2.
  • the first and second nucleotide in the 21 nucleotide sequence are exchanged for AC. These 21 nucleotide sequence are listed in Table 1. 13.
  • an output report is generated. In this report, the cDNA sequence is graphically represented, together with all the designed 21 nucleotides sequence, and with the similar and dissimilar EST sequences. In a second part of the report, the designed 21 nucleotide sequences are listed with the characteristics mentioned in point 1 to 7.
  • siRNA expression constructs Non viral siRNA expression constructs :
  • siRNA expression constructs The construction of the siRNA expression constructs is depicted in figure 1.
  • oligos containing knock-down target sequences as depicted in table 1 are cloned in the knock-down vector, pKD122.
  • This non-viral DNA expression plasmids can be used 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.
  • the siRNA expression construct can also so be contained in viruses.
  • the viruses can be made in an arrayed format, if preferred.
  • the arrayed viruses mediate expression of the siRNA constructs; each well contains a unique recombinant virus carrying a siRNA expression construct targeted against a gene, i.e. one target gene per well. Further details about the concept of arrayed adenoviral vectors can be found in WO 9964582, US 6,340,595 and 6,413,776 (Arrayed adenoviral libraries for performing functional genomics) .
  • pKD122 two other materials are needed for the generation of recombinant adenovirus particles: a helper cosmid and a packaging cell line (see also W09964582. US 6,340,595).
  • the cosmid (pWE/Ad.AflII-rITR ⁇ E2A) 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 (RCAs) are prepared.
  • 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 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. Design of oligos:
  • Oligonucleotides are designed to be targeted against specific mRNAs .
  • 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 forming a duplexed structure that is used 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:
  • N21 are DNA sequences corresponding to the sequences as depicted in Table 1, N21* is the antisense sequence of this sequence .
  • the single stranded oligonucleotide components are synthesized and annealed in 96 or 384 well plates to generate the double stranded oligonucleotides at a final concentration of 50 pmol/ ⁇ l, 100 ⁇ l total volume per well (Sigma) .
  • the knock-down expression vector, pKD122 (figure 3) is based on the pIPspAdapt ⁇ (WO 9964582) .
  • the pIPspAdApt ⁇ plasmids contain the 5' part (bp 1-454 and bp 3511-6093 of the adenovirus serotype 5 genome in which the El gene is deleted and a promoter is introduced.
  • the siRNA expression vectors of pIPspAdapt lack the CMV promoter and the SV40 polyadenylation site and the larger part of the polylinker, pKD122 further contains U6 promoter, Sap I recognition sites and the E.Coli lethal gene, ccdB .
  • Sap I cuts adjacent to its recognition sites (GCTCTTC (N) ⁇ / ) creating a 3' overhang (see figure 2). This has the advantage that it cuts any sequence containing the recognition sequence but the recognition sequence will not be present in the final construct since as it will be present on the excised fragment.
  • Sapl is used for the construction of expression plasmids.
  • the ccdB is included in the fragment to be excised. When the restriction fragment is not correctly excised and the ccdB gene remains in the plasmid, after transfection no E. coli colonies are formed. Only E. coli containing correct expression plasmids with the two unique Sapl overhangs and without the ccdB gene will form colonies.
  • pIPspAdapt6 was grown in the methylase negative E. coli strain DM1 to prevent methylation of the second Xba-site.
  • the DNA was isolated and digested with Xba I, thereby excising a 142 bp fragment containing the poly A signal.
  • the religated vector is called pIPspAdapt6-deltaPolyA.
  • the polylinker was removed from pIPspAdapt6-deltaPolyA by digestion with EcoRI and BamHl, blunted with Klenow, religated and digested with Ascl to reduce background This religated vector is called pIPspAdapt6-deltaPolyA delta- polylinker.
  • pIPspAdapt ⁇ -deltaPolyA-delta polylinker was digested with Avrll and Hindlll to remove the CMV promoter and purified on a 1% agarose TAE gel and isolated using the Qiaquick gel extraction kit (Qiagen) .
  • the ccdB gene is cut from pIPspAdaptlOZeoDestA
  • the genomic human U6 gene (Accession number M14486 (GenBank, NCBI) ) is cloned by a PCR based strategy using human genomic DNA. The region to be cloned starts at nucleotide -265 upstream of the transcription start site until nucleotide +198 downstream of the transcription start site.
  • the primers used are:
  • the PCR product is cloned into the Xba I and Hind III sites of pIPspAdapt ⁇ -deltaPolyA, the resulting vector is hU6(+l) pIPspAdapt6-dpA.
  • the digested R- and L-fragments together with the digested pIPspAdapt6-deltaPolyA delta polylinker and the ccdB fragment are ligated with T4 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: 11889 and 11890 this should generate a 1000 bp fragment.
  • Positive clones are digested with Hindi and BgLII, the correct clones give fragments of 3800, 1400, 538, 402 and 134 bp in size. Clones that give these fragments are sequenced.
  • the resulting vector is pKD122 (figure 3) Cloning of the oligos
  • the knockdown vector pKD122 (figure 3) is digested by
  • Ligation of the annealed oligos in the knock-down vector 0.5 ⁇ l digested knock-down vector (40 ng/ ⁇ l) , 1 ⁇ l T4 DNA ligase buffer (lOx concentrated, Biolabs) 0.5 ⁇ l T4 DNA ligase (Biolabs) and 7 ⁇ l milliQ are added per well. Added to this is 1 ⁇ l of the diluted annealed oligos. The plates are incubated over night at RT. Transformation : 5 ⁇ l of each ligation mix is put into a new PCR plate and put on ice 25 ⁇ l competent DH5 ⁇ -cells (Subcloning efficiency, Invitrogen) is 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. 170 ⁇ l RT SOC-medium (Invitrogen) is added to each well. The bacteria are recovered by shaking for 1 hour at 37 °C and 100-150 rpm.
  • 3 colonies of each construct are picked and inoculated as agar-stab (LB agar with 100 ⁇ g/ml ampicillin) and liquid culture (LB medium with 100 ⁇ g/ml ampicillin) .
  • the clones in the agar-stab are sequenced. Clones with the correct sequence are transfered to a new 96 well plate.
  • the knock-down vector with the annealed oligos is digested with PI-PspI.
  • the mixture is incubated over night at 65°C in a humified box.
  • the final virus propagation step is aimed at obtaining a higher percentage of wells showing CPE 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.
  • a 96 channel Hydra dispenser Robots
  • 20 ⁇ l of the supernatant is transferred onto PER.C6/E2A cells seeded in 96 well plates at a density of 2.25xl0 4 cells/well in 180 ⁇ l of DMEM + 10% FBS.
  • Cells are incubated at 34°C, 10% C0 2 for approximately 10 days and the number of wells showing CPE is scored. In general, the number of wells showing CPE is increased after propagation.
  • the plates are then stored at - 80°C.
  • 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.
  • GNAS encodes the G ⁇ subunit of G s , a heterotrimeric G- protein ( ⁇ , ⁇ , ⁇ ) .
  • G proteins interact with 7 transmembrane receptors, the so-called G-protein coupled receptor (GPCR) . Binding of a ligand to the GPCR induces a conformational change of the receptor that results in activation of the G- protein.
  • GPCR G-protein coupled receptor
  • the activated G protein will dissociate into its ⁇ subunit and the ⁇ subunit.
  • the ⁇ subunit will interact with adenylate cyclase and in turn activate this enzyme.
  • Adenylate cyclase converts ATP into cAMP.
  • cAMP responsive elements CRE
  • CREBP activated cAMP responsive element binding protein
  • PKA Protein Kinase A
  • the following assay is designed to measure the knockdown of GNAS at a functional level.
  • U20S cells are infected with an adenoviral construct encoding a GPCR ( ⁇ 2 adrenergic receptor) that couples to G s (MOI 500) together with an adenoviral reporter construct carrying CRE elements upstream of a luciferase gene (MOI 750) .
  • the infected cells are co-infected with either an adenoviral construct encoding a RNA molecule (SEQ ID NO: 1342) targeted against GNAS or an empty virus (MOI 1500) .
  • the ⁇ 2 adrenergic receptor is activated with isoproterenol, which is an agonist for the ⁇ 2 adrenergic receptor.
  • the luciferase activity is measured.
  • successful knock-down of GNAS results in a much lower cAMP levels compared with cAMP levels observed in the control cells transfected with empty virus. o ensure that the decreased cAMP levels are due to the knock-down of GNAS and not due to a non-specific effect on cAMP levels, forskolin is added to the triply infected cells. Forskolin increases intracellular cAMP levels by direct activation of adenylate cyclase and therefore is independent of G-protein (GNAS) .
  • GNAS G-protein
  • Figure 4 shows the results of the functional knock-down measurements of GNAS.
  • the luciferase activity in cells infected with the adenoviral GNAS knock-down construct are much lower than the luciferase levels of the cells that are not infected with the GNAS knock-down construct.
  • the forskolin results show that the cAMP reduction is due to the knock-down of GNAS rather then a general down-stream effect on the signal transduction route leading to the activation of CREBP .
  • Syk (NM_003177) is a tyrosine kinase that is essential for mast cell degranulation mediated by IgE.
  • IgE a tyrosine kinase that is essential for mast cell degranulation mediated by IgE.
  • Moriya et al 1997) it is shown that a specific inhibitor abolished IgE-mediated mast cell degranulation. If a knockdown construct against Syk is successful then mast cells are unable to degranulate.
  • Mast cell degranulation can be determined by measuring the release of ⁇ -hexosaminidase .
  • Total hexosaminidase is measured from the hydrolysis of the synthetic substrate 4- methylumbelliferyl N-acetyl- ⁇ -D-glucosaminide by hexosaminidase, which releases fluorescent 4- methylumbelliferone .
  • Experimental Culture of human Mast cells (hMC's)
  • Human mast cells are derived from cord blood mononuclear cells (#2C-150A; IO 8 cells/vial, Cambrex) .
  • Cells are cultured in RPMI 1640 (#52400-025, Gibco BRL) supplemented with 10% FBSHI (Gibco BRL), 1% MEM non essential amino acids (#11140- 35, Gibco BRL), 1% L-Glutamin (200mM, lOOx) (#25030-024,
  • the cell suspensions are seeded at a density of IO 6 cells/ml and the entire volume of cytokine supplemented medium is replaced on a weekly basis.
  • the adherent fraction of cells is discarded weekly by the transfer of the non- adherent cells to fresh culture flasks.
  • Cells are cultured for 9-11 weeks before use as described by Ochi et al (1999) Two weeks prior to the experiment cells are cultured with addition of 10 ng/ml hIL-4 and 5 ⁇ g/ml human myeloma IgE (Biodesign) to enhance Fc ⁇ RI expression as described by Ochi et al (2000) .
  • Cells are checked by toluidine blue staining and Flow cytometric analysis. Toluidine blue staining is performed on a weekly basis starting 6 th week of culture. Cells are stained for C-kit and FceRI expression and analyzed by FACS.
  • Cells are seeded at a density of 1E+05 cells/well in a 96 well V-bottom plate (Greiner) and transduced with an Adenovirus at a MOI of 1000.
  • Three days past infection the culture medium containing virus is removed from the cells and replaced by 200 ⁇ l of 1500 ng/ml anti-IgE (#501, DAKO) in culture medium. After one-hour of stimulation, at 37 °C, 100 ⁇ l supernatant is transferred to a (black) flat-bottom 96 well plate (Greiner) .
  • SykT402 a dominant negative form of Syk, which is a key player in the degranulation pathway, as shown by Moriya et al (1997) .
  • SykT402 is a truncated form of WT Syk at amino acid 402. Cells with the SykT402 mutant are unable to degranulate.
  • cells are transduced, at a MOI of 1000.
  • Cells are transduced either with the SykT402 mutant, a Syk knock-down virus construct (SEQ ID NO: 1338 and see example 2) or empty virus as a negative control.
  • Knock-down virus directed against GL2 is taken along to check for specific knock-down.
  • Three days past transduction cells are stimulated with 1500 ng/ml anti-IgE as described in degranulation assay' . Without stimulation the mast cells have already a ⁇ -hexosaminidase release of ⁇ 7%, this background is subtracted from all values measured.
  • anti-IgE only no viral infection
  • empty virus
  • the functional effect of an adenoviral knock-down was studied using the well-known NFKB pathway by measuring the phosphorylation of I B ⁇ , one of the inhibitory proteins which normally associate with and hold NF- ⁇ B inactive in the cytoplasm.
  • I B ⁇ one of the inhibitory proteins which normally associate with and hold NF- ⁇ B inactive in the cytoplasm.
  • Activation of the IKK kinase complex by TNF ⁇ stimulation results in phosphorylatation of I ⁇ B ⁇ , followed by its ubiquitination and degradation by the 26S proteosome. Phosphorylation and degradation of I ⁇ B ⁇ releases NF- ⁇ B, allowing it to translocate into the nucleus to activate target genes.
  • the phosphorylation state of I ⁇ B ⁇ was determined by using the Bio-Plex phospho-I ⁇ B ⁇ kit (Bio-Rad) , an assay that is similar to a capture sandwich ELISA but utilizes antibodies covalently coupled to dyed polystyrene beads.
  • U20S cells were infected with adenoviral knock-downs as indicated. Six days post infection, cell lysates were prepared after either treatment with 50 ng/mL of TNF ⁇
  • Bio-Plex phospho-IiB ⁇ assays were performed according to the manufacturer's instruction. Briefly, equal amount of cellular proteins were incubated overnight with the dyed beads conjugated with the I ⁇ B ⁇ capture antibodies. After a series of washes to remove unbound protein, a biotinylated detection antibody specific for Phospho-I ⁇ B ⁇ (Ser32) was added to the reaction. Streptavidin-phycoerythrin (PE) was added to detect the formation of a sandwich of antibodies around the phosphoraylated I ⁇ B ⁇ . The Luminex IS100 system was used to read each individual bead in the reaction mixture (Luminex, Austin) .
  • the constitutively expressed matrix metalloproteinase MMP2 was targeted for knock-down with 2 different adenoviral constructs (Ad-siRNA- MMP2-1 (SEQ ID NO: 633 or Ad-siRNA-MMP2-3 SEQ ID NO: 635) in primary human synoviocytes .
  • Ad-siRNA- MMP2-1 SEQ ID NO: 633 or Ad-siRNA-MMP2-3 SEQ ID NO: 635
  • Supematants of primary synoviocytes were analysed 6 days after infection at MOI 7500 with Ad-siRNA-MMP2-l and Ad-siRNA-MMP2-3, and compared to Ad- siRNA-control, a virus containing a target sequence against M6PR.
  • Yoshiuchi T Miyamoto M, Yamada K. ER-27319, an acridone- related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of fcepsilon receptor I-mediated activation of Syk. Proc Natl Acad Sci U S A. 1997, 94 (23) : 12539-44.
  • Wilson JM Grossman M, Wu CH, Chowdhury NR, Wu GY, Chowdhury JR. Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits. J Biol Chem. 1992, 267 (2) :963-7.
  • Table 1 Knock-down target sequences, 21 consecutive nucleotides.
  • the target gene is given in column 1 and 2. Accession numbers starting with SK:are from the kinase database (Manning et al, 2002), ENS from ENSEMBL database and other from GenBank.
  • NM_000858 GUK1 10 ACATCAAGGCCACCGATCTGC
  • NM_030662 MAP2K2 24 ACTTCGAAAGGATCTCAGAGC
  • NM_001323 CST6 46 ACTTTGAGGTCCTTGTGGTTC
  • NM_003505 FZD1 158 ACCATCGTCATCGCCTGCTAC
  • NM_005281 GPR3 190 ACGGACCTATGTGATGCTGGC
  • NM_005913 MC5R 214 ACCTACGTCATCCTGTGCCTC
  • NM_006056 GPR66 220 ACGCTACTGTTTGAGATGGTC
  • NM_012344 NTSR2 227 ACCGCGCTCCAAGTCTTTATC
  • NM_031866 FZD8 262 ACCGTGGTCTTCTTGCTGGTC
  • NM_001394 DUSP4 309 ACGGCTCTGTTGAATGTCTCC
  • NM_057158 DUSP4 309 ACGGCTCTGTTGAATGTCTCC
  • NM_001394 DUSP4 310 ACCTCGCAGTTCGTCTTCAGC
  • NM_004178 TARBP2 336 ACGCCTGTGTACGACCTTCTC
  • NM_134323 TARBP2 336 ACGCCTGTGTACGACCTTCTC
  • NM_134324 TARBP2 336 ACGCCTGTGTACGACCTTCTC
  • NM_001150 ANPEP 351 ACCTGAAGAAGCAGGTCACAC
  • NM_000129 F13A1 369 ACCAAGGATTCAGTGTGGAAC
  • NM_000133 F9 371 ACAACCATGACATTGCCCTTC
  • NM_000133 F9 372 ACATGTTCTGTGCTGGCTTCC
  • NM_000133 F9 373 ACCAAGGTATCCCGGTATGTC
  • NM_001908 CTSB 380 ACGGCTGTAATGGTGGCTATC
  • NM_005143 HP 500 ACCATAGCTGAGAACTAATGC
  • NM_004413 DPEP1 502 ACGTCCTGAGGCTGGTGAAAC
  • NM_005110 GFPT2 520 ACACCTGTGTTCAGGGATGAC
  • NM_005040 PRCP 522 ACATCGATACTATGGAGAGTC
  • NM_005468 NAALADASEL 525 ACAACCCTATGCTGCCTATGC
  • NM_004131 GZMB 545 ACGAAACAATGGCATGCCTCC
  • NM_006290 TNFAIP3 569 ACAGCGTCCAGGTTCCAGAAC
  • AF073344 AF073344 576 ACTGCCATTTGTGCCACAGGC NM_006537 USP3 576 ACTGCCATTTGTGCCACAGGC
  • AF073344 AF073344 577 ACAGCAGGAAGGCGGACATAC NM_006537 USP3 577 ACAGCAGGAAGGCGGACATAC
  • NM_021612 ADAM 11 617 ACCAGCAACTTTGCCAAGTCC
  • NM_014244 ADAMTS2 624 ACCTGCACTCCTATGACTGCC
  • NM_003183 ADAM 17 625 ACCAGAGAATGGACACCATCC
  • NM_021832 ADAM 17 625 ACCAGAGAATGGACACCATCC
  • NM_004994 MMP9 640 ACGGCAATGCTGATGGGAAAC
  • NM_021251 CAPN10 648 ACCTACAAGGTTGTGCCCTCC
  • NM_003474 ADAM 12 652 ACAATCCCAGGAGATTGCTGC
  • IMMP2L 656 ACACCGTGGTGACATTGTATC
  • J04605 HUMPEPD 676 ACGTCACAGAAGCCCTCTGTC NM_000285 PEPD 676 ACGTCACAGAAGCCCTCTGTC
  • NM_023038 ADAM 19 682 ACCTGGAGAAGAATGAGCAAC
  • NM_033274 ADAM 19 682 ACCTGGAGAAGAATGAGCAAC
  • NM_023038 ADAM 19 684 ACTGGAACCAAGTGTGGCTAC
  • NM_003814 ADAM20 695 ACTTCTCCAGAAGTGGTGATC
  • NM_014296 CAPN7 700 ACAATAGTATCGGATTGCTCC
  • COL6A3 710 ACCAGAGTAAGCCTGAGATCC
  • COL6A3 712 ACATCGACAGAACAGAGCTGC
  • NM_057165 COL6A3 712 ACATCGACAGAACAGAGCTGC
  • NM_057166 COL6A3 712 ACATCGACAGAACAGAGCTGC
  • NM_057167 COL6A3 712 ACATCGACAGAACAGAGCTGC
  • NM_021627 SENP2 728 ACAGAAGAACGGACGATCTCC

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Abstract

La présente invention a trait à des polynucléotides comportant une séquence d'ARN comprenant une première suite de 21 nucléotides consécutifs et une deuxième suite de 21 nucléotides consécutifs, complémentaire à la première suite de 21 nucléotides consécutifs, où la première suite de nucléotides consécutifs: dans la direction 5'→ 3', le premier nucléotide est un nucléotide A, le deuxième nucléotide est un nucléotide C et le dernier nucléotide est un nucléotide C ; et aucune suite d'au moins quatre nucléotides consécutifs identiques n'est présente, le nombre total de nucléotides G et C est compris entre 33 et 71 % du nombre total de nucléotides dans la direction 5'→ 3', les nucléotides 3 à 21 sont homologues à la molécule ARN. L'invention a également trait à des vecteurs codant pour les nucléotides de la présente invention. En outre, l'invention à trait à des bibliothèques de polynucléotides et de vecteurs codant pour les polynucléotides et des procédés de production de vecteurs et de bibliothèques.
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CN118320133B (zh) * 2024-06-14 2024-09-24 中国医学科学院基础医学研究所 一种重组腺相关病毒在治疗心力衰竭中的应用
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CN119570927A (zh) * 2024-09-25 2025-03-07 温州医科大学附属第一医院 一种wsb1作为靶点在hph疾病中的应用

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