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US 2025/0051845 A1Published application
Method and kit for diagnosis, prognosis, therapy monitoring or outcome prediction assessment of a disease
AssigneeImmagina Biotechnology S.r.l.
InventorsAlessia DEL PIANO, Tea KECMAN, Massimiliano CLAMER
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Abstract
An in vitro or ex vivo method of diagnosis, prognosis, therapy monitoring or outcome prediction assessment of a disease or condition in a subject by detecting and quantifying at least one molecular marker of the disease or condition contained in a biological sample of the subject, wherein the at least one molecular marker is an RNA fragment comprising a 3′ phosphate or a 2′/3′ cyclic phosphate and kit for diagnosing Spinal Muscular Atrophy and cutaneous Squamous Cell Carcinoma.
Claims12
An in vitro or ex vivo method of diagnosis, prognosis, therapy monitoring or outcome prediction assessment of a disease or condition in a subject by detecting and quantifying at least one molecular marker of the disease or condition contained in a biological sample of the subject, wherein the at least one molecular marker is an RNA fragment comprising a 3′ phosphate or a 2′/3′ cyclic phosphate (3′P RNA), the method comprising the following steps:
- (a′) phosphorylating the 5′ end of the at least one 3′P RNA contained in the biological sample and obtaining at least one phosphorylated RNA fragment;
- (b′) ligating the 3′ end of the at least one phosphorylated RNA fragment to the 5′ end of an RNA-based adapter obtaining at least one first ligation product, wherein the RNA-based adapter has formula (Ia):
- 5′OH-E1-Az-E2-OH3′ (Ia)
- wherein
- E1 is a first oligoribonucleotide sequence having a length comprised between 15 and 30,
- A is an abasic site or a spacer allowing the arrest of a retrotranscriptase enzyme activity,
- z is an integer number from 1 to 5,
- E2 is a second oligoribonucleotide sequence having a length comprised between 15 and 30;
- (c′) self-ligating the at least one first ligation product to form at least one circular RNA molecule;
- (d′) performing a reverse transcription of the at least one circular RNA molecule obtaining at least one single strand cDNA molecule comprising the sequence of the at least one 3′P RNA, wherein the reverse transcription is carried out using a reverse transcription primer having formula (IIa):
- 5′OH-G-F1-OH3′ (IIa)
- wherein
- F1 is the reverse complement deoxyoligoribonucleotide of E1, wherein complementarity of F1 to E1 is comprised between 60% and 100%, and
- G is a first DNA oligonucleotide having a length comprised between 10 and 30;
- (e′) performing in parallel a first and a second qPCR amplifications of the at least one single strand cDNA molecule obtaining a first and a second amplification product, wherein:
- the first qPCR amplification is carried out using a first pair of primers annealing on at least one part of the 3′P RNA sequence, the first pair of primers being selected from pairs of primer Set1, Set2 and Set3, wherein each pair of primers comprises a forward and reverse primer, wherein the forward and reverse primers of the pair of primers Set1 have a sequence as set forth in formulas (IIIa) and (IVa), respectively, the forward and reverse primers of the pair of primers Set2 have a sequence as set forth in formulas (IIIa′) and (IVa′), respectively, and the forward and reverse primers of the pair of primers Set3 have a sequence as set forth in formulas (IIIa″) and (IVa″), respectively:
- Set1
- 5′ OH-H2-N—OH 3′ (IIIa)
- 5′ OH-H1-M-OH 3′ (IVa)
- Set2
- 5′ OH-H2-N—OH 3′ (IIIa′)
- 5′ OH-H1-OH 3′ (IVa′)
- Set3
- 5′OH-H2-OH3′ (IIIa″)
- 5′OH-H1-M-OH3′ (IVa″)
- wherein
- H1 is a second DNA oligonucleotide annealing on E1, wherein complementarity of H1 to E1 is comprised between 30% and 100%, and
- M is a third DNA oligonucleotide having a length comprised between 6 and 30 and annealing on at least 6 nucleotides of the 3′ end of the 3′P RNA sequence,
- H2 is a fourth DNA oligonucleotide annealing on E2, wherein complementarity of H2 to E2 is comprised between 30% and 100%, and
- N is a fifth DNA oligonucleotide having a length comprised between 6 and 30 and annealing on at least 6 nucleotides of the 5′ end of the 3′P RNA sequence;
- the second qPCR amplification is carried out using a second pair of primers annealing on the RNA-based adapter sequence, the second pair of primers comprising a second forward and a second reverse primer having formula (Va) and (VIa), respectively:
- 5′OH-H2-OH3′ (Va)
- 5′OH-11-OH3′ (VIa)
- wherein
- H2 has the meaning set forth above, and
- I1 is a sixth DNA oligonucleotide annealing on G, wherein complementarity of I1 to G is comprised between 60% and 100%;
- and
- (f) repeating steps (a′) to (e′) on at least one control sample, wherein the control sample is a biological sample of a healthy, treated or non-treated subject;
- (g′) determining Ct values for the at least one 3′P RNA and for the RNA-based adapter in the first and second amplification products for either the biological sample and the control sample,
- (h′) calculating a 3′P-qPCR fold change of the Ct values determined in step (g′) according to the following equation:
- 3
- ′
-
- P
-
- qPCR
-
- fold
-
- change
- =
- 2
- Ct
-
- (
- B
- )
- 3
- ′
-
- P
- -
- Ct
-
- (
- A
- )
- 3
- ′
-
- P
- 2
- Ct
-
- (
- B
- )
- adp
- -
- Ct
-
- (
- A
- )
- adp
- =
- 2
- Δ
-
- Ct
-
- (
- 3
- ′
-
- P
- )
- 2
- Δ
-
- Ct
-
- (
- adp
- )
- =
- 2
- ΔΔ
-
- Ct
- wherein
- Ct(A)3′P is the Ct value for the 3′P RNA determined in the biological sample,
- Ct(B)3′P is the Ct value for the 3′P RNA determined in the control sample,
- Ct(A)adp is the Ct value for the RNA-based adapter determined in the biological sample,
- Ct(B)adp is the Ct value for the RNA-based adapter determined in the control sample;
- wherein a 3′P-qPCR fold change value≥2 or ≤0.5 is indicative of the diagnosis, prognosis, therapy monitoring or outcome prediction assessment of the disease or condition.
113 more elements
A kit for the diagnosis, prognosis, therapy monitoring or outcome prediction assessment of the Spinal Muscular Atrophy (SMA) comprising:
- (a′) an RNA-based adapter having a sequence as set forth in SEQ ID No.:
- 185;
- (b′) a reverse transcription primer having a sequence as set forth in SEQ ID No.:
- 186;
- (c′) seven first pairs of primers for performing a first qPCR amplification, the first forward and reverse primers of the seven primers pairs having a sequence as set forth in the following sequence pairs:
- SEQ ID No.:
- 187 and 188, SEQ ID No.:
- 187 and 189, SEQ ID No.:
- 190 and 191, SEQ ID No.:
- 192 and 193, and SEQ ID No.:
- 198 and 199, SEQ ID No.:
- 200 and 201, SEQ ID No.:
- 202 and 203;
- and
- (d′) a second pair of primers for performing a second qPCR amplification, the second forward and second reverse primers having a sequence as set forth in SEQ ID No.:
- 204 and 205, respectively.
13 more elements
A kit for the diagnosis, prognosis, therapy monitoring or outcome prediction assessment of cutaneous Squamous Cell Carcinoma (cSCC) comprising:
- (a′) an RNA-based adapter having a sequence as set forth in SEQ ID No.:
- 185,
- (b′) a reverse transcription primer having a sequence as set forth in SEQ ID No.:
- 186,
- (c′) four first pairs of primers for performing a first qPCR amplification, the first forward and reverse primers of the two primers pairs having respectively a sequence as set forth in the following sequence pairs:
- SEQ ID No.:
- 194 and 195, SEQ ID No.:
- 196 and 197, SEQ ID No.:
- 222 and 223, SEQ ID No.:
- 224 and 225, and
- (d′) a second pair of primers for performing a second qPCR amplification, the second forward and second reverse primers having a sequence as set forth in SEQ ID No.:
- 204 and 205, respectively.
9 more elements
Dependencies are inferred from the claim text.