Nothing Special   »   [go: up one dir, main page]

WO2024179870A1 - Plac8 antisense molecules used as a medicament - Google Patents

Plac8 antisense molecules used as a medicament Download PDF

Info

Publication number
WO2024179870A1
WO2024179870A1 PCT/EP2024/054144 EP2024054144W WO2024179870A1 WO 2024179870 A1 WO2024179870 A1 WO 2024179870A1 EP 2024054144 W EP2024054144 W EP 2024054144W WO 2024179870 A1 WO2024179870 A1 WO 2024179870A1
Authority
WO
WIPO (PCT)
Prior art keywords
plac8
antisense molecule
molecule according
overexpression
antisense
Prior art date
Application number
PCT/EP2024/054144
Other languages
French (fr)
Inventor
Thomas M. GRESS
Malte Buchholz
Original Assignee
Philipps-Universität Marburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philipps-Universität Marburg filed Critical Philipps-Universität Marburg
Publication of WO2024179870A1 publication Critical patent/WO2024179870A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/113—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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy

Definitions

  • the present invention relates to antisense molecules for use as a medicament , in particular the use of these antisense molecules for the treatment of diseases associated with the overexpression of PLAC8 , a method for treating disease associated with overexpression of PLAC8 and a kit containing these antisense molecules .
  • the antisense- mediated degradation of nuclear PLAC8 mRNA is described as a novel therapeutic approach for the treatment of mainly pancreatic malignancies . It has been found that pancreatic cancer cells are addicted to expression of the PLAC8 mRNA, but not the encoded protein, for maintenance of genome integrity and cell viability
  • pancreas pancreatic ductal adenocarcinoma ; PDAC
  • PDAC pancreatic ductal adenocarcinoma
  • chemotherapeutic approaches have predominantly been used, which unspeci f ically target rapidly proli ferating cells . Since this also af fects numerous normal cells within the body, such therapies are usually associated with severe undesired side ef fects .
  • Molecular-targeted therapies that speci fically address molecular changes in tumor cells and are therefore less harmful to healthy body cells have not yet been success fully established for PDAC therapy .
  • Another novel concept are so- called immune checkpoint inhibitor therapies , which have already shown impressive success in other tumor entities . They aim to inhibit mechanisms by which tumor cells protect themselves from attack by the body ' s own immune system .
  • These therapies have also proven largely inef fective in PDAC to date , but can presumably be rendered ef fective by generating DNA damage in tumor cells , since this dramatically increases the number of immune-stimulatory signals .
  • the technical problem underlying the present invention is to provide means for treating diseases associated with the overexpression of the protein PLAC8 avoiding the problems of the prior art approaches .
  • the present invention provides an antisense molecule binding to the mRNA of the protein PLAC8 for use as a medicament .
  • Antisense oligonucleotides are synthetic, short-chain, single-stranded nucleic acids that are made up of a few nucleotides ( typically from 15 up to 30 nucleotides ) from a freely selectable sequence of bases .
  • ASOs bind via complementary base pairing to a nucleic acid whose base sequence matches them ( opposite base pairs - " anti-sense” ) .
  • the antisense molecule is used for the treatment of diseases associated with an overexpression of PLAC8, for example cancer, like a pancreatic tumor, acute myeloide leukemia (AML) , cervical cancer, glioblastoma or renal clear cell carcinoma.
  • the pancreatic tumor can be a pancreatic ductal adenocarcinoma (PDAC) or a pancreatic neuroendocrine tumor (PanNET) .
  • the antisense molecule is an antisense oligonucleotide, which can have from 15 up to 30 nucleotides, in particular 15 to 20 nucleotides.
  • the antisense molecule can be selected from the group consisting of siRNA, shRNA and GapmeRs .
  • GapmeRs contain a central DNA region flanked on both sides with RNA regions. That is, the GapmeR can be represented in a simplified formula as RNA-DNA-RNA. That is, GapmeRs are DNA antisense structures with RNA-like segments on both sides of the sequence. These linear pieces of genetic information are designed to hybridize to a target piece of RNA (in the present case PLAC8) and silence the gene through the induction of cleavage. Binding of the GapmeRs to the target can have higher affinity due to modified RNA flanking regions, as well as resistance to degradation by nucleases.
  • the RNA regions can be chemically modified.
  • the chemical modification can be selected from the group consisting of 2'-0Me modified bases or 2'-F modified bases or a methylene bridge between the 2'-0 and the 4'-C-atom.
  • GapmeRs having such a chemical modification of the RNA regions are known as LNA GapmeRs (LNA means locked nucleic acids) , wherein the RNA is locked in an ideal Watson-Crick pairing conformation due to the locking provided by the chemical modification.
  • Antisense molecules used according to the present invention are known to the skilled person so that materials and methods for producing them are available . Furthermore , such antisense molecules are commercially available .
  • LNA Gampers can be purchased from Qiagen .
  • the antisense molecule can be used together with a so-called immune checkpoint inhibitor .
  • This use can be carried out in that the checkpoint inhibitor is delivered before , together with or after the delivery of the antisense molecule .
  • the antisense molecule as defined above for treating a disease associated with overexpression of PLAC8 as defined above as well .
  • a further subj ect-matter of the present invention is a method for treating a disease associated with overexpression of PLAC8 with an antisense molecule .
  • a disease associated with overexpression of PLAC8 with an antisense molecule .
  • the antisense molecule and the diseases it is referred to the above detailed description in its entirety .
  • the present invention relates to a kit containing an antisense molecule directed against the mRNA of PLAC8 and instructions for use for treating a disease associated with PLAC8 overexpression .
  • the kit further contains an immune checkpoint inhibitor .
  • PLAC8 is a gene encoding a small protein with unknown function .
  • PLAC8 is practically absent in normal pancreatic tissue , but is regularly found highly overexpressed in PDAC as well as in other tumors . It has previously been shown that the PLAC8 gene , which codes for an evolutionarily highly conserved small protein of 115 AA, is strongly ectopically expressed in pancreatic ductal adenocarcinoma ( PDAC ) as well as a subset of pancreatic neuroendocrine tumors ( PanNET ) . Inhibition of PLAC8 expression signi ficantly impairs cell proli feration and viability in PDAC and PanNET cells .
  • the functional entity mediating the cell-intrinsic pro-tumorigenic ef fects is not the PLAC8 protein, but the PLAC8 mRNA itsel f .
  • the functional entity mediating the cell-intrinsic pro-tumorigenic ef fects is not the PLAC8 protein, but the PLAC8 mRNA itsel f .
  • the mRNA itsel f that is critical for tumor cell survival and proli feration, speci fically in a function within the nucleus itsel f (by default , mRNAs are transported out of the nucleus and translated into protein at ribosomes ) .
  • LNA GapmeRs are in parts chemically modi fied DNA molecules , they are protected from degradation in the organism and can therefore also be used in vi vo .
  • Figure 1 shows data about PLAC8.
  • FIG. 1 shows the CRISPR/Cas9 targeting of PLAC8.
  • Figure 3 shows the genotype of S2-007-derived clone G2#l.
  • Figure 4 shows the pedigree of S2-007-derived clones.
  • Figure 5 shows the genomic organization and splice variants.
  • Figure 6 shows principles of function of the LNA-GapmeRs .
  • Figure 7 shows the GapmeRs effects in G2#l clone.
  • Figure 8 shows the effects of GapmeRs in G2#l clones.
  • the gene is located on chromosome 4 with 3-4 alleles per cell (S2 — 007 cell line) .
  • the gene codes for a small protein (18 kDa) highly conserved in evolution. It is overexpressed in PDAC, which leads to enhanced cell proliferation so that it can be considered to be an oncogene.
  • S2-007 clones with targeted inactivation of PLAC8 expression were generated using the CRISPR/Cas9 technology.
  • the successful inactivation of all alleles should have abrogated protein and RNA expression completely (integration of an antibiotics resistance/ transcription stop cassette) .
  • Clones devoid of PLAC8 protein were readily obtained, but grew as well as parental cells, and still reacted to PLAC8-specif ic siRNA transfection.
  • the S2-007 clone is deprived of PLAC8 protein expression generated using the CRISPR/Cas9 technology.
  • the usage of guide RNA 2 Clone G2#l was chosen for the CRISPR/Cas9 approach described in the following as this clone already had 2-3 incorporated transcription-stop cassettes at the PLAC8 gene locus and only one remaining "wt"-allele (with a frameshift caused by NHEJ) , which results in cells lacking the PLAC8 protein, but not the PLAC8 RNA.
  • the Clone G2#l derives from the S2-007 cell line and was created via a first round of CRISPR/Cas9 editing using a donor plasmid coding for a puromycin transcription-stop cassette.
  • genotype of clone G2#l it is already known that this clone has 3 integrated puromycin transcription-stop cassettes and 1 allele with a frameshift (4 NT-deletion) within exon 2 at the PLAC8 gene locus. This results in a gene KO at the protein, but not at the RNA level.
  • Fig. 4 shows the pedigree of S2-007-derived clones.
  • the second round only yielded clones with additional indels, but not a single clone without RNA expression.
  • LNA-GapmeRs The principle of function of LNA-GapmeRs is represented in Fig. 6.
  • GapmeR's in general have at least one of the following properties: They are antisense oligonucleotides (ASO, they are short (15-20 bases) single stranded nucleic acids. They have a central DNA "gap” (central DNA part) flanked by RNA pars on each side. They form DNA: RNA duplexes detected by RNaseH. Flanking regions contain " locked nucleic acids” ( LNA) which are nuclease resistant and have enhanced binding .
  • ASO antisense oligonucleotides
  • LNA locked nucleic acids
  • Fig . 7 shows individual/ independent GapmeRs elicited ef ficient knockdown of both, PLAC8 protein ( in parental cells ) and PLAC8 mRNA ( in PLAC8 protein-proficient as well as protein-deficient cell lines/clones ) .
  • PLAC8 knockdown led to profound growth inhibition regardless of protein status , far surpassing ef ficacy of siRNAs .
  • Fig . 8 it represents flow cytometry ( PI staining) showing slightly pronounced S-Phase , but BrdU assays demonstrated that cell proli feration was severely impaired after PLAC8 knockdown . Moreover, gamma-H2AX staining revealed induction on massive amounts of cell death, peaking 24h after trans fection of GapmeRs .
  • PLAC8 protein-deficient clones showed no di f ferences in growth rates compared to parental cell lines , but strongly reacted to PLAC8 mRNA knockdown, in particular to LNA GapmeR-mediated knockdown of nuclear-located PLAC8 mRNA, with induction of massive DNA damage and growth inhibition .
  • Nanopore sequencing identi fied several novel PLAC8 splice isoforms , but recombinant expression of none of these isoforms was able to rescue the knockdown phenotype .
  • PLAC8 mRNA i functions similarly to a cis-acting long non-coding RNA; ii ) has an important and non-redundant role in maintaining genomic integrity in pancreas cancer cells ; and ill ) functions completely independently of the encoded protein . Both aspects ( separate functions of mRNA and encoded protein, and a central role of an mRNA in maintaining genome stability) have not previously been described in any cellular context .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates to an antisense molecule binding to the mRNA of the protein PLAC8 for use as a medicament, for example for the treatment of cancer, in particular pancreatic ductal adenocarcinoma. Furthermore, the use of such antisense molecules and a method for treating of cancer as well as a kit containing the antisense molecules are described.

Description

PLAC8 Antisense Molecules Used as a Medicament
The present invention relates to antisense molecules for use as a medicament , in particular the use of these antisense molecules for the treatment of diseases associated with the overexpression of PLAC8 , a method for treating disease associated with overexpression of PLAC8 and a kit containing these antisense molecules . In the following the antisense- mediated degradation of nuclear PLAC8 mRNA is described as a novel therapeutic approach for the treatment of mainly pancreatic malignancies . It has been found that pancreatic cancer cells are addicted to expression of the PLAC8 mRNA, but not the encoded protein, for maintenance of genome integrity and cell viability
Malignant tumors of the pancreas (pancreatic ductal adenocarcinoma ; PDAC ) are extremely aggressive and presently incurable . Currently, available treatment options have very limited ef ficacy and are usually associated with severe side ef fects .
Overexpression of the PLAC8 gene in pancreatic tumors has been reported several times in the literature . Proli feration inhibition of pancreatic cancer cells in vi tro ( i . e . , in cell culture ) after downregulation of PLAC8 expression has also been described; however, other techniques were used in these cases that did not speci fically target nuclear mRNA; the ef fects observed should be improved; and speci fically, the ef fect on chromosome stability was not observable in these previous studies .
In the treatment of pancreatic cancer, chemotherapeutic approaches have predominantly been used, which unspeci f ically target rapidly proli ferating cells . Since this also af fects numerous normal cells within the body, such therapies are usually associated with severe undesired side ef fects . Molecular-targeted therapies that speci fically address molecular changes in tumor cells and are therefore less harmful to healthy body cells have not yet been success fully established for PDAC therapy . Another novel concept are so- called immune checkpoint inhibitor therapies , which have already shown impressive success in other tumor entities . They aim to inhibit mechanisms by which tumor cells protect themselves from attack by the body ' s own immune system . These therapies have also proven largely inef fective in PDAC to date , but can presumably be rendered ef fective by generating DNA damage in tumor cells , since this dramatically increases the number of immune-stimulatory signals .
The technical problem underlying the present invention is to provide means for treating diseases associated with the overexpression of the protein PLAC8 avoiding the problems of the prior art approaches .
This has been solved by the subj ect-matter of the independent claims . Preferred embodiments are defined in the dependent claims .
The present invention provides an antisense molecule binding to the mRNA of the protein PLAC8 for use as a medicament .
Antisense oligonucleotides ( or ASOs for short ) are synthetic, short-chain, single-stranded nucleic acids that are made up of a few nucleotides ( typically from 15 up to 30 nucleotides ) from a freely selectable sequence of bases . ASOs bind via complementary base pairing to a nucleic acid whose base sequence matches them ( opposite base pairs - " anti-sense" ) . In one embodiment, the antisense molecule is used for the treatment of diseases associated with an overexpression of PLAC8, for example cancer, like a pancreatic tumor, acute myeloide leukemia (AML) , cervical cancer, glioblastoma or renal clear cell carcinoma. The pancreatic tumor can be a pancreatic ductal adenocarcinoma (PDAC) or a pancreatic neuroendocrine tumor (PanNET) .
In one embodiment, the antisense molecule is an antisense oligonucleotide, which can have from 15 up to 30 nucleotides, in particular 15 to 20 nucleotides.
In another embodiment, the antisense molecule can be selected from the group consisting of siRNA, shRNA and GapmeRs . GapmeRs contain a central DNA region flanked on both sides with RNA regions. That is, the GapmeR can be represented in a simplified formula as RNA-DNA-RNA. That is, GapmeRs are DNA antisense structures with RNA-like segments on both sides of the sequence. These linear pieces of genetic information are designed to hybridize to a target piece of RNA (in the present case PLAC8) and silence the gene through the induction of cleavage. Binding of the GapmeRs to the target can have higher affinity due to modified RNA flanking regions, as well as resistance to degradation by nucleases.
Thus, in a further embodiment, the RNA regions can be chemically modified. For example, the chemical modification can be selected from the group consisting of 2'-0Me modified bases or 2'-F modified bases or a methylene bridge between the 2'-0 and the 4'-C-atom. GapmeRs having such a chemical modification of the RNA regions are known as LNA GapmeRs (LNA means locked nucleic acids) , wherein the RNA is locked in an ideal Watson-Crick pairing conformation due to the locking provided by the chemical modification. Antisense molecules used according to the present invention are known to the skilled person so that materials and methods for producing them are available . Furthermore , such antisense molecules are commercially available . For example , LNA Gampers can be purchased from Qiagen .
In a further embodiment , the antisense molecule can be used together with a so-called immune checkpoint inhibitor . This use can be carried out in that the checkpoint inhibitor is delivered before , together with or after the delivery of the antisense molecule .
According to the present invention, there is further provided the use of the antisense molecule as defined above for treating a disease associated with overexpression of PLAC8 as defined above as well .
A further subj ect-matter of the present invention is a method for treating a disease associated with overexpression of PLAC8 with an antisense molecule . For the details of such a method, in particular the antisense molecule and the diseases , it is referred to the above detailed description in its entirety .
Moreover, the present invention relates to a kit containing an antisense molecule directed against the mRNA of PLAC8 and instructions for use for treating a disease associated with PLAC8 overexpression .
In one embodiment , the kit further contains an immune checkpoint inhibitor .
PLAC8 is a gene encoding a small protein with unknown function . PLAC8 is practically absent in normal pancreatic tissue , but is regularly found highly overexpressed in PDAC as well as in other tumors . It has previously been shown that the PLAC8 gene , which codes for an evolutionarily highly conserved small protein of 115 AA, is strongly ectopically expressed in pancreatic ductal adenocarcinoma ( PDAC ) as well as a subset of pancreatic neuroendocrine tumors ( PanNET ) . Inhibition of PLAC8 expression signi ficantly impairs cell proli feration and viability in PDAC and PanNET cells .
One new aspect is that the functional entity mediating the cell-intrinsic pro-tumorigenic ef fects is not the PLAC8 protein, but the PLAC8 mRNA itsel f . To be more speci fic, it has been demonstrated through extensive experiments that , unexpectedly, it is not the protein, but the mRNA itsel f that is critical for tumor cell survival and proli feration, speci fically in a function within the nucleus itsel f (by default , mRNAs are transported out of the nucleus and translated into protein at ribosomes ) . Using commercially available antisense molecules ( ' LNA GapmeRs ' ; purchased from Qiagen GmbH) , it has been shown that targeted degradation of PLAC8 mRNA in the nucleus leads to massive DNA damage in the cell and, as a consequence , to complete inhibition of proli feration and ultimately cell death . Since LNA GapmeRs are in parts chemically modi fied DNA molecules , they are protected from degradation in the organism and can therefore also be used in vi vo .
Based on the above finding, it has therefore been concluded that targeting nuclear PLAC8 mRNA in tumor cells , e . g . , using nuclear antisense DNA molecules , would be a promising novel approach for pancreatic cancer therapy as well as other tumors . In this context , the ef fect of severe DNA damage is of particular interest as it is known from the literature that DNA damage contributes to the formation of ' neo-antigens ' , which make tumor cells visible to the immune system and can improve the effectiveness of so-called checkpoint inhibitor therapies, which have not yet shown efficacy in e.g. PDAC therapy. Moreover, the same principle could also be applied to other types of tumors which regularly show strong overexpression of PLAC8, such as acute myeloid leukemia (AML) , cervical cancer, glioblastoma, renal clear cell carcinoma etc.
The present invention will be further illustrated by the following examples and figures. It is explicitly pointed out that the examples shall not be construed to limit the invention thereto. The examples are only intended to illustrate the invention.
Figure 1 shows data about PLAC8.
Figure 2 shows the CRISPR/Cas9 targeting of PLAC8.
Figure 3 shows the genotype of S2-007-derived clone G2#l.
Figure 4 shows the pedigree of S2-007-derived clones.
Figure 5 shows the genomic organization and splice variants.
Figure 6 shows principles of function of the LNA-GapmeRs .
Figure 7 shows the GapmeRs effects in G2#l clone.
Figure 8 shows the effects of GapmeRs in G2#l clones.
Examples
Methods
Nanopore sequencing, CRISPR-mediated genome editing, LNA- GapmeR-mediated gene silencing, fluorescence microscopy, flow cytometry, FISH/chromosome painting, SNP array analyses Fig.l shows the previously known data relating to FLAG 8. The gene is located on chromosome 4 with 3-4 alleles per cell (S2 — 007 cell line) . The gene codes for a small protein (18 kDa) highly conserved in evolution. It is overexpressed in PDAC, which leads to enhanced cell proliferation so that it can be considered to be an oncogene.
As to Fig. 2, S2-007 clones with targeted inactivation of PLAC8 expression were generated using the CRISPR/Cas9 technology. The successful inactivation of all alleles should have abrogated protein and RNA expression completely (integration of an antibiotics resistance/ transcription stop cassette) . Clones devoid of PLAC8 protein were readily obtained, but grew as well as parental cells, and still reacted to PLAC8-specif ic siRNA transfection.
As shown in Fig. 3, the S2-007 clone is deprived of PLAC8 protein expression generated using the CRISPR/Cas9 technology. The usage of guide RNA 2 Clone G2#l was chosen for the CRISPR/Cas9 approach described in the following as this clone already had 2-3 incorporated transcription-stop cassettes at the PLAC8 gene locus and only one remaining "wt"-allele (with a frameshift caused by NHEJ) , which results in cells lacking the PLAC8 protein, but not the PLAC8 RNA.
The Clone G2#l derives from the S2-007 cell line and was created via a first round of CRISPR/Cas9 editing using a donor plasmid coding for a puromycin transcription-stop cassette. With regard to the genotype of clone G2#l it is already known that this clone has 3 integrated puromycin transcription-stop cassettes and 1 allele with a frameshift (4 NT-deletion) within exon 2 at the PLAC8 gene locus. This results in a gene KO at the protein, but not at the RNA level. Fig. 4 shows the pedigree of S2-007-derived clones. Second round of CRISPR/Cas9 editing in G2#l cells using a donor plasmid coding for a neomycin transcription-stop cassette and GuideRNA 1 is shown. The hypothetical genotype of these clones would consist of 3 integrated puromycin transcription-stop cassettes and 1 integrated neomycin transcription-stop cassette within exon 2 at the PLAC8 gene locus. This would result in a PLAC8-K0 at the protein and RNA level due to integrated transcriptional stop signals (polyA-signals ) into all of the four alleles at the PLAC8 gene locus.
However, the second round only yielded clones with additional indels, but not a single clone without RNA expression.
All in all, more than 250 clones were analyzed so far following different CRISPR/Cas-rounds ; no PLAC8 RNA-def icient clone was ever observed so that PLAC8 mRNA is relevant, not the protein.
Genomic organization of the PLAC8 gene locus on chromosome 4 and splice variants in pancreatic cancer cells as determined by Nanopore sequencing and RACE PGR (see Fig. 5) .
The principle of function of LNA-GapmeRs is represented in Fig. 6. Mechanism: Rnase H ubiquitous in almost all organisms for degradation of DNA- RNA- Hybrids (e.g. virus defence) GapmeR binds mRNA so that the degradation of the hybrid by RNaseH is achieved, which in turn results in complete degradation of the target mRNA within the cell nucleus.
GapmeR's in general have at least one of the following properties: They are antisense oligonucleotides (ASO, they are short (15-20 bases) single stranded nucleic acids. They have a central DNA "gap" (central DNA part) flanked by RNA pars on each side. They form DNA: RNA duplexes detected by RNaseH. Flanking regions contain " locked nucleic acids" ( LNA) which are nuclease resistant and have enhanced binding .
Fig . 7 shows individual/ independent GapmeRs elicited ef ficient knockdown of both, PLAC8 protein ( in parental cells ) and PLAC8 mRNA ( in PLAC8 protein-proficient as well as protein-deficient cell lines/clones ) . PLAC8 knockdown led to profound growth inhibition regardless of protein status , far surpassing ef ficacy of siRNAs .
As to Fig . 8 , it represents flow cytometry ( PI staining) showing slightly pronounced S-Phase , but BrdU assays demonstrated that cell proli feration was severely impaired after PLAC8 knockdown . Moreover, gamma-H2AX staining revealed induction on massive amounts of cell death, peaking 24h after trans fection of GapmeRs .
Results
CRISPR/Cas genome editing approaches aimed at complete abrogation of PLAC8 mRNA expression (homologous repair templates with transcription termination elements ) consistently failed to inactivate all PLAC8 alleles in cancer cells , even in repeat rounds using di f ferent selectable markers . Clones deficient in producing the PLAC8 protein, however, were readily obtained ( random indel mutations disrupting the PLAC8 ORF following non-homologous end j oining) . Moreover, PLAC8 protein-deficient clones showed no di f ferences in growth rates compared to parental cell lines , but strongly reacted to PLAC8 mRNA knockdown, in particular to LNA GapmeR-mediated knockdown of nuclear-located PLAC8 mRNA, with induction of massive DNA damage and growth inhibition . Nanopore sequencing identi fied several novel PLAC8 splice isoforms , but recombinant expression of none of these isoforms was able to rescue the knockdown phenotype . Conclusion
The results imply that the PLAC8 mRNA i ) functions similarly to a cis-acting long non-coding RNA; ii ) has an important and non-redundant role in maintaining genomic integrity in pancreas cancer cells ; and ill ) functions completely independently of the encoded protein . Both aspects ( separate functions of mRNA and encoded protein, and a central role of an mRNA in maintaining genome stability) have not previously been described in any cellular context .

Claims

Claims
1. Antisense molecule binding to the mRNA of the protein PLAC8 for use as a medicament.
2. Antisense molecule according to claim 1 for use in the treatment of diseases associated with an overexpression of PLAC8.
3. Antisense molecule according to claim 2, wherein the disease associated with the overexpression of PLAC8 is cancer.
4. Antisense molecule according to claim 3, wherein the cancer is a pancreatic tumor, acute myeloide leukemia (AML) , cervical cancer, glioblastoma or renal clear cell carcinoma.
5. Antisense molecule according to claim 4, wherein the pancreatic tumor is pancreatic ductal adenocarcinoma (PDAC) or pancreatic neuroendocrina tumor (PanNET) .
6. Antisense molecule according to any of the preceding claims, wherein the antisense molecule is an antisense oligonucleotide .
7. Antisense molecule according to claim 6, wherein the oligonucleotide has 15 to 30 nucleotides.
8. Antisense molecule according to claim 6 or 7, wherein the antisense molecule is selected from the group consisting of siRNA, shRNA and GapmeRs .
9. Antisense molecule according to claim 8, wherein the RNA regions are chemically modified.
10. Antisense molecule according to claim 9, wherein the chemical modification is selected from the group consisting of 2'-OMe modified bases, 2'-F modified bases and a methylene bridge between the 2'-0 and the 4'-C-atom.
11. Antisense molecule according to any of the preceding claims, wherein further an immune checkpoint inhibitor is used .
12. Use of the antisense molecule as defined in any of claims 1 and 6 to 10 for treating a disease associated with an overexpression of PLAC8 as defined in any of claims 1 to 5.
13. Method for treating a disease associated with an overexpression of PLAC8 with an antisense molecule.
14. Kit containing an antisense molecule directed against the mRNA of PLAC8 and instructions for use for treating a disease associated with an PLAC8 overexpression.
15. Kit according to claim 1 further containing a checkpoint inhibitor .
PCT/EP2024/054144 2023-03-01 2024-02-19 Plac8 antisense molecules used as a medicament WO2024179870A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23159355 2023-03-01
EP23159355.9 2023-03-01

Publications (1)

Publication Number Publication Date
WO2024179870A1 true WO2024179870A1 (en) 2024-09-06

Family

ID=85415392

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/054144 WO2024179870A1 (en) 2023-03-01 2024-02-19 Plac8 antisense molecules used as a medicament

Country Status (1)

Country Link
WO (1) WO2024179870A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009045443A2 (en) * 2007-10-02 2009-04-09 The University Of Rochester Methods and compositions related to synergistic responses to oncogenic mutations
WO2020139897A1 (en) * 2018-12-27 2020-07-02 Sirnaomics, Inc. Silencing tgf-beta 1 and cox2 using sirnas delivered in combination with immune checkpoint inhibitors to treat cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009045443A2 (en) * 2007-10-02 2009-04-09 The University Of Rochester Methods and compositions related to synergistic responses to oncogenic mutations
WO2020139897A1 (en) * 2018-12-27 2020-07-02 Sirnaomics, Inc. Silencing tgf-beta 1 and cox2 using sirnas delivered in combination with immune checkpoint inhibitors to treat cancer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRAJESH P. KAISTHA: "PLAC8 Localizes to the Inner Plasma Membrane of Pancreatic Cancer Cells and Regulates Cell Growth and Disease Progression through Critical Cell-Cycle Regulatory Pathways", CANCER RESEARCH, vol. 76, no. 1, 1 January 2016 (2016-01-01), US, pages 96 - 107, XP093153017, ISSN: 0008-5472, Retrieved from the Internet <URL:https://aacrjournals.org/cancerres/article-pdf/76/1/96/2730997/96.pdf> DOI: 10.1158/0008-5472.CAN-15-0216 *
HUANG MAO-LING ET AL: "Placenta specific 8 gene induces epithelial-mesenchymal transition of nasopharyngeal carcinoma cells via the TGF-[beta]/Smad pathway", EXPERIMENTAL CELL RESEARCH, vol. 374, no. 1, 26 November 2018 (2018-11-26), pages 172 - 180, XP085556936, ISSN: 0014-4827, DOI: 10.1016/J.YEXCR.2018.11.021 *
MISHA MAO: "Multifaced roles of PLAC8 in cancer", BIOMARKER RESEARCH, vol. 9, no. 1, 9 October 2021 (2021-10-09), London, UK, XP093153525, ISSN: 2050-7771, Retrieved from the Internet <URL:https://link.springer.com/article/10.1186/s40364-021-00329-1/fulltext.html> DOI: 10.1186/s40364-021-00329-1 *
SHI LIPING ET AL: "Overexpression of placenta specific 8 is associated with malignant progression and poor prognosis of clear cell renal cell carcinoma", INTERNATIONAL UROLOGY AND NEPHROLOGY, AKADEMIAI, BUDAPEST, HU, vol. 49, no. 7, 27 March 2017 (2017-03-27), pages 1165 - 1176, XP036259107, ISSN: 0301-1623, [retrieved on 20170327], DOI: 10.1007/S11255-017-1578-Y *

Similar Documents

Publication Publication Date Title
US10900036B2 (en) RNA interactome of polycomb repressive complex 1 (PRC1)
Brysch et al. Design and application of antisense oligonucleotides in cell culture, in vivo, and as therapeutic agents
CN111575372B (en) Long non-coding RNA LETN as tumor marker and treatment target
CA2515243A1 (en) Agent for inhibiting development or progress of proliferative diseases and especially cancer diseases and pharmaceutical composition containing said agent
KR101052289B1 (en) Treatment of melanoma with a decrease in the amount of cholesterol
US20160024597A1 (en) miRNAs AS THERAPEUTIC TARGETS IN CANCER
US20190078090A1 (en) Downregulating mir-132 for the treatment of lipid related disorders
JP7406278B2 (en) Long non-coding RNA LETN as a tumor marker and therapeutic target
WO2007149521A2 (en) Nucleotide motifs providing localization elements and methods of use
Maksimenko et al. Oncogene-targeted antisense oligonucleotides for the treatment of Ewing sarcoma
WO2008099396A1 (en) Use of h19-silencing nucleic acid agents for treating restenosis
Weirauch et al. U1 adaptors for the therapeutic knockdown of the oncogene pim-1 kinase in glioblastoma
WO2024179870A1 (en) Plac8 antisense molecules used as a medicament
Fluiter et al. Killing cancer by targeting genes that cancer cells have lost: allele-specific inhibition, a novel approach to the treatment of genetic disorders
EP2336320B1 (en) Sensizitation of cancer cells to therapy using sina targeting genes from the 1p and 19q chromosomal regions
Marchelli et al. Gene modulation by peptide nucleic acids (PNAs) targeting microRNAs (miRs)
Prins et al. Antisense of oligonucleotides and the inhibition of oncogene expression
JP2022541212A (en) SiRNA sequences targeting the expression of the human genes JAK1 or JAK3 for therapeutic use
KR102483859B1 (en) Guide RNA for deleting MYC enhancer and use thereof
US11357853B2 (en) Inhibition of a lncRNA for treatment of neuroblastoma
JP2011188849A (en) miR-7 EXPRESSION PLASMID HAVING ANTITUMOR EFFECT
WO2012131673A2 (en) Ccat-1 silencing nucleic acid agents for treating cancer
EP3999644A2 (en) Inhibitors of microrna 451a for treatment of endometriosis
KR20240034143A (en) Novel genomic safe harbor and the use thereof
WO2019246571A1 (en) Compositions and methods for treating adrenocortical carcinoma

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24706690

Country of ref document: EP

Kind code of ref document: A1