EP-3765631-B1 - METHODS FOR THE NON-INVASIVE DETECTION AND MONITORING OF THERAPEUTIC NUCLEIC ACID CONSTRUCTS

EP3765631B1EP 3765631 B1EP3765631 B1EP 3765631B1EP-3765631-B1

Inventors

ODEGAARD, Justin I.
SIKORA, MARCIN
ARTSIOMENKA, Aliaksandr

Dates

Publication Date: 20260506
Application Date: 20190313

Claims (14)

A method of detecting a presence or absence of a therapeutic nucleic acid construct in a biological sample from a test subject at least partially using a computer, the method comprising: receiving, by the computer, test sequence information comprising sequence reads obtained from cell-free nucleic acid molecules in the biological sample; removing, by the computer, one or more of the sequence reads that originate from one or more intronic regions and/or from one or more regions spanning exon-intron junctions from the test sequence information to generate filtered test sequence information; or identifying, by the computer, one or more of the sequence reads that originate from one or more regions spanning exon-exon junctions from the test sequence information to generate enriched test sequence information; or identifying, by the computer, at least one set of the sequence reads comprising at least one locus in common with one another, which set comprises a coverage that exceeds a threshold value to thereby identify a candidate set of sequence reads; and, identifying, by the computer, one or more of the sequence reads in the filtered or the enriched test sequence information, or at least some members of the candidate set of sequence reads, that substantially align with differentiating reference sequence information that originates from the therapeutic nucleic acid construct, thereby detecting the presence or absence of the therapeutic nucleic acid construct in the biological sample from the test subject.
The method of claim 1, wherein the method comprises differentiating reference sequence information that originates from a vector portion of the therapeutic nucleic acid construct.
The method of claim 1 or claim 2, wherein a plurality of said sequence reads are present in a unimodal size distribution.
The method of any one of the preceding claims, wherein the biological sample is selected from the group consisting of: blood, plasma, and serum.
The method of any one of the preceding claims, further comprising generating the test sequence information from the cell-free nucleic acid molecules in the biological sample; and/or amplifying one or more segments of the cell-free nucleic acid molecules in the biological sample to generate amplified nucleic acid molecules, optionally further comprising sequencing the amplified nucleic acid molecule to generate the test sequence information.
The method of any one of the preceding claims, (i) wherein the test sequence information is obtained from targeted sequences of the cell-free nucleic acid molecules in the biological sample, wherein the targeted sequences are obtained by selectively enriching one or more regions from the cell-free nucleic acid molecules in the biological sample prior to sequencing, the method optionally further comprising amplifying the obtained targeted sequences prior to sequencing; (ii) further comprising attaching one or more adapters comprising barcodes to the cell-free nucleic acid molecules prior to sequencing; and/or (iii) wherein the sequencing is selected from the group consisting of: targeted sequencing, intron sequencing, exome sequencing, and whole genome sequencing.
The method of any one of the preceding claims, wherein the test subject is a mammalian subject, for example wherein the mammalian subject is a human subject, optionally wherein the test subject has a disease or disorder capable of being treated with the therapeutic nucleic acid construct, for example wherein (i) the disease is cancer; (ii) the disorder is a genetic disorder; (iii) the disease is a viral disease; or (iv) the disease or disorder is selected from the group consisting of immune deficiency disorders, hemophilia, thalassemia, sickle cell disease, blood disease, chronic granulomatous disorder, congenital blindness, lysosomal storage disease, muscular dystrophy, cancer, neurodegenerative disease, viral infections, bacterial infections, epidermolysis bullosa, heart disease, fat metabolism disorder, and diabetes, or a combination thereof.
The method of any one of the preceding claims, wherein the therapeutic nucleic acid construct (i) is a construct used in a DNA-based therapy, for example wherein the DNA-based therapeutic construct is selected from the group consisting of plasmids, aptamers, DNAzymes, antisense oligonucleotides, viral vectors, and antigene oligonucleotides; (ii) comprises a natural nucleotide sequence, a variant of the natural nucleotide sequence or a synthetic nucleotide sequence or combinations thereof; (iii) is part of a chimeric antigen receptor (CAR) T-cell therapy; (iv) is part of a CRISPR/Cas therapy; or (v) comprises a portion of a human adenovirus nucleic acid sequence.
The method of any one of the preceding claims, wherein the therapeutic nucleic acid construct is a construct used in an RNA-based therapy, for example wherein the RNA-based therapeutic nucleic acid construct is selected from the group consisting of aptamers, RNA decoys, antisense RNA, ribozymes, small interfering RNAs, and microRNA.
The method of any one of the preceding claims, wherein the biological sample comprises fragments of the therapeutic nucleic acid construct, the method optionally further comprising selectively enriching for a target sequence of the therapeutic nucleic acid construct, for example wherein the target sequence comprises (i) a natural nucleotide sequence, a variant of the natural nucleotide sequence or a synthetic nucleotide sequence or combinations thereof; or (ii) a gene or a variant thereof selected from the group consisting of TP53, HBB, RPE65, and B-domain deleted factor VIII gene, DMD, UL123, APOB, SMN1, SMN2, ICAM-1, TLR9, IRS-1, VEGF, PDGFA, PDGFB, PDGFC, PDGFD, and TTR.
The method of any one of the preceding claims, further comprising using results of the method as an input to generate a report, for example wherein the report provides an indication of the presence or absence of the therapeutic nucleic acid construct in the biological sample from the test subject.
A system comprising means for carrying out the method of claim 1.
A computer program comprising instructions which, when the program is executed by a computer, cause the computer to carry out the method of claim 1.
A computer-readable storage medium comprising instructions which, when executed by a computer, cause the computer to carry out the method of claim 1.

Description

BACKGROUND Cell-free nucleic acids (cfNA) are utilized for genomic alteration detection to inform therapy selection and is an emerging non-invasive disease monitoring tool. Therapeutic nucleic acids (TNAs) directly or indirectly manipulate DNA or mRNA transcript levels for therapeutic effect. While cfNA is in routine clinical use and TNAs are in an expanding number of clinical trials, little is known about potential interactions of these technologies, including the need for cfNA next generation sequencing (NGS) to recognize this source of exogenous DNA or RNA. Like natural nucleic acids, nucleic acid constructs used in TNAs may be shed into the peripheral blood and may be found in the acellular fraction (i.e., not in intact cells). This material may be extracted and sequenced, and the synthetic constructs may be detected through unique synthetic sequences or over-representation of natural sequences. This detection and quantitation may be used in a number of ways, including to ascertain efficacy of administration, persistence or biological efficacy of synthetic constructs, or efficacy of therapy. However, such detection methods for TNAs in samples comprising cell-free nucleic acids are not yet established. Baoutina et al. (Hum Gene Ther Methods. 2013; 24(6):345-54) discuss "Improved detection of transgene and nonviral vectors in blood" (title). SUMMARY The invention is set out in the appended set of claims. References to methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body are to be read as products, e.g. substances or compositions, for use in such methods. In certain aspects, the present disclosure relates to a method of detecting a presence of a therapeutic nucleic acid construct in a biological sample from a test subject at least partially using a computer, the method comprising receiving, by the computer, test sequence information comprising sequence reads obtained from cell-free nucleic acid molecules in the biological sample; removing, by the computer, one or more of the sequence reads that originate from one or more intronic regions and/or from one or more regions spanning exon-intron junctions from the test sequence information to generate filtered test sequence information; and, identifying, by the computer, one or more of the sequence reads in the filtered test sequence information that substantially align with differentiating reference sequence information that originates from the therapeutic nucleic acid construct, thereby detecting the presence of the therapeutic nucleic acid construct in the biological sample from the test subject. In certain aspects, the present disclosure relates to a method of detecting a presence of a therapeutic nucleic acid construct in a biological sample from a test subject at least partially using a computer, the method comprising receiving, by the computer, test sequence information comprising sequence reads obtained from cell-free nucleic acid molecules in the biological sample; identifying, by the computer, one or more of the sequence reads that originate from one or more regions spanning exon-exon junctions from the test sequence information to generate enriched test sequence information; and, identifying, by the computer, a presence of one or more of the sequence reads in the enriched test sequence information that substantially align with differentiating reference sequence information that originates from the therapeutic nucleic acid construct, thereby detecting the presence of the therapeutic nucleic acid construct in the biological sample from the test subject. In certain aspects, the present disclosure relates to a method of detecting a of therapeutic nucleic acid construct in a biological sample from a test subject at least partially using a computer, the method comprising: receiving, by the computer, test sequence information comprising sequence reads obtained from cell-free nucleic acid molecules in the biological sample; identifying, by the computer, at least one set of the sequence reads comprising at least one locus in common with one another, which set comprises a coverage that exceeds a threshold value to thereby identify a candidate set of sequence reads; and, identifying, by the computer, at least some members of the candidate set of sequence reads that substantially align with reference sequence information that originates from the therapeutic nucleic acid construct, thereby detecting the presence of the therapeutic nucleic acid construct in the biological sample from the test subject. A therapeutic nucleic acid construct can be administered to the test subject prior to receiving the test sequence information. Monitoring therapeutic efficacy in a test subject can be performed at least partially using a computer by: receiving, by the computer, test sequence information comprising sequence reads obtained from cell-free nucleic acid molecules in biological samples from the