Liposomal preparations for non-invasive-prenatal or cancer screening

Abstract

Disclosed are controls for use in identifying any of a number of genotypes and/or for use in identifying or characterizing a disease or condition. The controls may be particularly useful for diagnostic tests that utilize circulating cell-free DNA or microRNA. A control may comprise a mixture of nucleic acids. In some embodiments, a control comprises liposomes, e.g., wherein nucleic acids of the control are associated with the liposomes. For example, the controls are useful for determining whether a fetus comprises an aneuploidy. Also disclosed are methods of using the controls.

1 - 78 . (canceled) 
     
     
         79 . A control for use in identifying a plurality of genotypes, comprising liposomes and a first mixture of nucleic acids, wherein the first mixture of nucleic acids comprises a first plurality of nucleotide sequences, each nucleotide sequence of the first plurality encodes a genotype of the plurality of genotypes; each genotype of the plurality of genotypes is associated with a neoplasm; and the first mixture of nucleic acids is incorporated into the liposomes. 
     
     
         80 - 93 . (canceled) 
     
     
         94 . The control of  claim 79 , further comprising a second mixture of nucleic acids, wherein the second mixture of nucleic acids comprises a second plurality of nucleotide sequences, each nucleotide sequence of the second plurality is an alternate genotype that occurs at the same genetic locus as a nucleotide sequence of the first plurality; the second mixture of nucleic acids is admixed with the first mixture of nucleic acids; and the admixture of the first mixture of nucleic acids and the second mixture of nucleic acids is incorporated into the liposomes. 
     
     
         95 . The control of  claim 94 , wherein the second mixture of nucleic acids:
 a) encodes substantially all of a human genome and/or   b) consists of circulating, cell-free DNA obtained from a human donor.   
     
     
         96 . The control of  claim 95 , wherein the human genome:
 (a) is a peripheral blood mononuclear cell genome, lymphocyte genome, fibroblast genome, or adipocyte genome; or   b) is the genome of a human cell line; or   c) wherein the human cell line is GM24385.   
     
     
         97 - 99 . (canceled) 
     
     
         100 . The control of  claim 79 , wherein each nucleic acid of the first mixture of nucleic acids comprises:
 a) exactly one nucleotide sequence of the plurality of nucleotide sequences;   b) at least one nucleic acid that comprises more than one nucleotide sequence of the plurality of nucleotide sequences; and/or   c) multiple copies of a nucleic acid that comprises each nucleotide sequence of the plurality of nucleotide sequences.   
     
     
         101 - 102 . (canceled) 
     
     
         103 . The control of  claim 94 , wherein:
 a) the copy number of a nucleotide sequence comprising a genotype in the first mixture relative to the sum of (1) the copy number of the nucleotide sequence comprising the genotype in the first mixture and (2) the copy number of a nucleotide sequence comprising the alternate genotype in the second mixture is about 0.01% to about 30%, such as about 0.1% to about 10%; or   b) the copy number of any nucleotide sequence comprising a genotype in the first mixture relative to the sum of (1) the copy number of the nucleotide sequence comprising the genotype in the first mixture and (2) the copy number of a nucleotide sequence comprising the alternate genotype in the second mixture is about 0.01% to about 30%, such as about 0.1% to about 10%.   
     
     
         104 - 106 . (canceled) 
     
     
         107 . The control of  claim 79 , wherein:
 a) each nucleotide sequence of the first plurality of nucleotide sequences has a concentration of about 1 copy per mL to about 10 9  copies per mL in the control; or   b) each nucleotide sequence of the first plurality of nucleotide sequences has a concentration of about 10 copies per mL to about 106 copies per mL in the control; and/or   c) the nucleic acids in the first mixture make up about 5% to about 15% of the total concentration of nucleic acids in the control.   
     
     
         108 - 109 . (canceled) 
     
     
         110 . The control of  claim 79 , wherein:
 a) the median length of the nucleic acids in the control is about 35 base pairs to about 1000 base pairs; or   b) the median length of the nucleic acids in the control is about 50 base pairs to about 500 base pairs.   
     
     
         111 - 140 . (canceled) 
     
     
         141 . The control of  claim 79 , further comprising blood plasma or blood serum. 
     
     
         142 . The control of  claim 141 , wherein the blood plasma or blood serum is human blood plasma or human blood serum. 
     
     
         143 . The control of  claim 141 , wherein:
 a) about 50% to 100% of the control comprises blood plasma or blood serum;   b) about 90% to 99.999% of the control comprises blood plasma or blood serum; or   c) about 95% to 99.99% of the control comprises blood plasma or blood serum.   
     
     
         144 - 148 . (canceled) 
     
     
         149 . The control of  claim 79 , wherein the liposomes comprise:
 a) phospholipids; and/or   b) at least one lipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin and/or   c) at least one lipid selected from the group consisting of dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, dimyristoyl phosphatidylserine, distearoyl phosphatidylserine, dipalmitoyl phosphatidylserine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, distearoyl phosphatidylinositol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; and/or   d) at least one lipid selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (“DPPC”) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (“DMPC”); and/or   e) cholesterol.   
     
     
         150 - 158 . (canceled) 
     
     
         159 . The control of  claim 79 , wherein:
 a) the liposomes comprise a pegylated lipid; and/or   b) the liposomes comprise a pegylated lipid that is a PEG-modified phosphoethanolamine-based lipid; and/or   c) the pegylated lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350]; and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350].   
     
     
         160 . The control of  claim 79 , wherein:
 a) the median diameter of the liposomes is about 30 nm to about 1000 nm; and/or   b) the average diameter of the liposomes is about 100 nm to about 300 nm; and/or   c) the nucleic acids of the control are more stable than nucleic acids of a control that does not comprise liposomes; and/or   d) the nucleic acids of the control are more stable when stored at a temperature of about 0° C. to about 100° C. than nucleic acids of a control that does not comprise liposomes; and/or   e) the nucleic acids of the control are more stable when stored for a period of time of about 1 day to about 5 years than nucleic acids of a control that does not comprise liposomes; and/or   f) the nucleic acids of the control do not form aggregates when stored at a temperature of about 0° C. to about 100° C.; and/or   g) wherein the nucleic acids of the control do not form aggregates when stored for a period of time of about 1 day to about 5 years; and/or   h) the nucleic acids of the control are stable when stored for a period of time of at least about 4 months at a temperature of about 4° C. to about 50° C.; and/or   i) the nucleic acids of the control are stable when stored for a period of time of about 4 months to about 5 months at a temperature of about 4° C. to about 50° C.; and/or   j) the nucleic acids of the control do not form aggregates when stored for a period of time of at least about 4 months at a temperature of about 4° C. to about 50° C.; and/or   k) the nucleic acids of the control do not form aggregates when stored for a period of time of about 4 months to about 5 months at a temperature of about 4° C. to about 50° C.   
     
     
         161 - 205 . (canceled) 
     
     
         206 . The control of  claim 79 , wherein each genotype of the plurality of genotypes consists of a mutation:
 a) to a gene selected from the group consisting of ABL1, AKT1, AKT2, AKT3, ALK, APC, AR, AR1D1A, ARAF, ARHGAP5, ATM, ATR, BCL2, BCR, BRAF, BRC42, BRCA1, BRCA2, BRIP1, CBFB, CCND1, CCND2, CCNE1, CDH1, CDK4, CDK6, CDKN2A, CDKN2B, CSF1R, CTNNB1, DDR2, DNMT3A, EGFR, ERBB2 (“HER2”), ERBB3, ERBB4 (“HER4”), ERCC1, ESR1, ETV1, ETV4, ETV6, EWSR1, EZH2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GABRA6, GABRG2, GATA3, GNA11, GNAQ, GNAS, HNF1A, HPAS, HRAS, IDH1, IDH2, IHD2, JAK2, JAK3, KDR, KIT, KMT2A, KRAS, MAP2K1, MAP2K2, MECOM, MET, MKL1, MLH1, MLL, MPL, MSH2, MSH6, MTOR, MYC, MYCN, MYD88, NF1, NF2, NFE2L2, NOTCH1, NPM1, NRAS/CSDE1, NTRK1, NUP214, PALB2, PARP1, PARP2, PDGFRA, PDGFRB, PICALM, PIK3CA, PMS2, PTCH1, PTEN, PTPN11, RAD51, RAFT, RARA, RB1, RET, RHEB, RHOA, RIT1, ROS1, RUNX1, RUNX1T1, SMAD4, SMARCB1, SMO, SRC, STK11, TAL1, TCF3, TERT, TMPRSS2, TP53, TSC1, TSC2, and VHL; or   b) to a gene selected from the group consisting of MTOR, MPL, NRAS, PARP1, AKT3, DNMT3A, MSH2, IDH1, VHL, MLH1, MYD88, CTNNB1, ATR, PIK3CA, FGFR3, PDGFRA, KIT, FBXW7, APC, GABRG2, NPM1, EGFR, MET, BRAF, EZH2, JAK2, GNAQ, RET, PTEN, ATM, KRAS, PTPN11, FLT3, RB1, PARP2, ARHGAP5, AKT1, RAD51, IDH2, TP53, NF1, SMAD4, AKT2, ERCC1, and GNAS; or   c) to a gene selected from the group consisting of AKT1, ATM, BRAF, CDKN2A, CSF1R, EGFR, ERBB2 (“HER2”), ERBB4 (“HER4”), FGFR1, FGFR2, FGFR3, GNA11, HRAS, JAK2, JAK3, KDR, KIT, KRAS, MET, NOTCH1, NRAS, PDGFRA, PIK3CA, PTEN, RET, and STK11; or   d) to a gene selected from the group consisting of ABL1, AKT1, ALK, APC, AR, AR1D1A, ARAF, ATM, BCL2, BCR, BRAF, BRC42, BRCA1, BRCA2, BRIP1, CCND1, CCND2, CCNE1, CDH1, CDK4, CDK6, CDKN2A, CDKN2B, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, ETV1, ETV4, ETV6, EWSR1, EZH2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HER/ERBB2, HNF1A, HPAS, HRAS, IDH1, IDH2, IHD2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MAP2K2, MET, MLH1, MLL, MPL, MSH2, MSH6, MTOR, MYC, MYCN, NF1, NF2, NFE2L2, NOTCH1, NPM1, NRAS, NTRK1, PALB2, PDGFRA, PDGFRB, PIK3CA, PMS2, PTCH1, PTEN, PTPN11, RAFT, RARA, RB1, RET, RHEB, RHOA, RIT1, ROS1, SMAD4, SMARCB1, SMO, SRC, STK11, TERT, TMPRSS2, TP53, TSC1, TSC2, and VHL; or   e) to a gene selected from the group consisting of BRAF, EGFR, ERBB2, and KRAS; or   f) selected from the group consisting of mutation V600E to gene BRAF, mutation T790M to gene EGFR, mutation delL747-P753insS to gene EGFR, mutation A775_G776insYVMA to gene ERBB2, and mutation G12D to gene KRAS; or   g) to a gene selected from the group consisting of BRAF, EGFR, ERBB2, KIT, KRAS, NRAS, and PIK3CA; or   h) selected from the group consisting of mutation V600E to gene BRAF, mutation D770_N771insG to gene EGFR, mutation E746_A750delELREA to gene EGFR, mutation T790M to gene EGFR, mutation D816V to gene KIT, mutation G12D to gene KRAS, mutation Q61R to gene NRAS, mutation H1047R to gene PIK3CA, and mutation N1068fs*4 to gene PIK3CA; or   i) to a gene selected from the group consisting of AKT1, AKT2, AKT3, APC, ARHGAP5, ATM, ATR, BRAF, CTNNB1, DNMT3A, EGFR, ERBB2, ERCC1, EZH2, FBXW7, FGFR3, FLT3, GABRA6, GABRG2, GNAQ, GNAS, IDH1, IDH2, JAK2, KIT, KRAS, MET, MLH1, MPL, MSH2, MTOR, MYD88, NF1, NPM1, NRAS/CSDE1, PARP1, PARP2, PDGFRA, PIK3CA, PTEN, PTPN11, RAD51, RB1, RET, SMAD4, TP53, and VHL; or   j) selected from the group consisting of mutation 145G>A to gene AKT1, mutation c.49G>A to gene AKT1, mutation c.268G>T to gene AKT2, mutation c.371A>T to gene AKT3, mutation c.4248delC to gene APC, mutation c.4348C>T to gene APC, mutation c.4666_4667 insA to gene APC, mutation c.1864G>A to gene ARHGAP5, mutation c.1058_1059 delGT to gene ATM, mutation c.5557G>A to gene ATM, mutation c.3790_3796 delATAAAAG to gene ATR, mutation c.1799T>A to gene BRAF, mutation c.121A>G to gene CTNNB1, mutation c.2644C>T to gene DNMT3A, mutation c.2236_2250 de115 to gene EGFR, mutation c.2310_2311 insGGT to gene EGFR, mutation c.2369C>T to gene EGFR, mutation c.2573T>G to gene EGFR, mutation c.2324_2325 ins12 to gene ERBB2, mutation c.287C>A to gene ERCC1, mutation c.1937A>T to gene EZH2, mutation c.1394G>A to gene FBXW7, mutation c.746C>G to gene FGFR3, mutation c.2503G>T to gene FLT3, mutation c.763G>C to gene GABRA6, mutation c.1355A>G to gene GABRG2, mutation c.626A>C to gene GNAQ, mutation c.601C>T to gene GNAS, mutation c.394C>T to gene IDH1, mutation c.515G>A to gene IDH2, mutation c.419G>A to gene IDH2, mutation c.1849G>T to gene JAK2, mutation c.2447A>T to gene KIT, mutation c.1679T>A to gene KIT, mutation c.35G>A to gene KRAS, mutation c.3757T>G to gene MET, mutation c.1151T>A to gene MLH1, mutation c.1544G>T to gene MPL, mutation c.2250de1G to gene MSH2, mutation c.2359_2360 de1CT to gene MSH2, mutation c.2664A>T to gene MTOR, mutation c.794T>C to gene MYD88, mutation c.2987_2988 insAC to gene NF1, mutation c.4084C>T to gene NF1, mutation c.7501de1G to gene NF1, mutation c.863_864 insTCTG to gene NPM1, mutation c.182A>G to gene NRAS, mutation c.2738de1G to gene PARP1, mutation c.398A>C to gene PARP2, mutation c.1694_1695 insA to gene PDGFRA, mutation c.2525A>T to gene PDGFRA, mutation c.1633G>A to gene PIK3CA, mutation c.3140A>G to gene PIK3CA, mutation c.3204_3205 insA to gene PIK3CA, mutation c.388C>T to gene PTEN, mutation c.741_742 insA to gene PTEN, mutation c.800de1A to gene PTEN, mutation c.226G>A to gene PTPN11, mutation c.433C>T to gene RAD51, mutation c.958C>T to gene RB1, mutation c.2753T>C to gene RET, mutation c.1394_1395 insT to gene SMAD4, mutation c.818G>A to gene TP53, mutation c.743G>A to gene TP53, mutation c.723delC to gene TP53, mutation c.524G>A to gene TP53, mutation c.263delC to gene TP53, and mutation c.426_429 delTGAC to gene VHL; or   k) to a gene selected from the group consisting of BRAF, CTNNB1, EGFR, ERBB2, IDH1, KIT, KRAS, NRAS/CSDE1, PDGFRA, PIK3CA, PTEN, RET, and TP53.   l) selected from the group consisting of mutation c.1799T>A to gene BRAF, mutation c.121A>G to gene CTNNB1, mutation c.2236_2250 de115 to gene EGFR, mutation c.2369C>T to gene EGFR, mutation c.2573T>G to gene EGFR, mutation c.2324_2325 ins12 to gene ERBB2, mutation c.394C>T to gene IDH1, mutation c.1679T>A to gene KIT, mutation c.35G>A to gene KRAS, mutation c.182A>G to gene NRAS/CSDE1, mutation c.2525A>T to gene PDGFRA, mutation c.1633G>A to gene PIK3CA, mutation c.3140A>G to gene PIK3CA, mutation c.800delA to gene PTEN, mutation c.2753T>C to gene RET, and mutation c.524G>A to gene TP53.   
     
     
         207 . The control of claim  206 (f) wherein the plurality of genotypes comprises mutation V600E to gene BRAF, mutation T790M to gene EGFR, mutation delL747-P753insS to gene EGFR, mutation A775_G776insYVMA to gene ERBB2, and mutation G12D to gene KRAS. 
     
     
         208 . The control of claim  206 (h) wherein the plurality of genotypes comprises mutation V600E to gene BRAF, mutation D770_N771insG to gene EGFR, mutation E746_A750delELREA to gene EGFR, mutation T790M to gene EGFR, mutation D816V to gene KIT, mutation G12D to gene KRAS, mutation Q61R to gene NRAS, mutation H1047R to gene PIK3CA, and mutation N1068fs*4 to gene PIK3CA.