Microneedle devices and methods, and skin condition assays

Abstract

The invention is directed to reliable and efficient detection of mRNAs as well as other RNAs in living cells and its use to identify and, if desired, separate cells based on their desired characteristics Such methods greatly simplify and reduce the time necessary to carry out previously-known procedures, and offers new approaches as well, such as selecting cells that generate a particular protein or antisense oligonucleotide, generating cell lines that express multiple proteins, generating cell lines with knock-out of one or more protein, and others.

1 . A method of treating a subject with an autoimmune disease of the skin comprising:
 a. collecting a sample comprising RNA derived from skin from the subject, wherein the subject has not been administered an autoimmune therapeutic drug within 7 days prior to the collecting the sample comprising RNA;   b. determining an expression level of at least one gene based on the RNA;   c. predicting that a subject with the autoimmune disease of the skin will be responsive to the autoimmune therapeutic drug with a positive predictive value greater than 80% based on the expression level of the at least one gene; and   d. based on the predicting in (c), treating the subject with the autoimmune therapeutic drug.   
     
     
         2 . The method in  claim 1 , wherein the PPV is greater than 95% for a cohort that has greater than 100 patients. 
     
     
         3 . The method of  claim 1 , wherein the autoimmune therapeutic drug comprises an antibody. 
     
     
         4 . The method of  claim 1 , wherein the autoimmune therapeutic drug is an IL-17 mediated treatment, an IL-23 mediated treatment, or a TNFα mediated treatment. 
     
     
         5 . The method of  claim 1 , wherein the autoimmune therapeutic drug is at least one drug selected from the group consisting of: etanercept, infliximab, adalimumab, certolizumab, ustekinumab, secukinumab, ixekizumab, brodalumab, guselkumab, tildrakizumab, and risankizumab. 
     
     
         6 . The method of  claim 1 , wherein the at least one gene comprises at least five genes. 
     
     
         7 . The method of  claim 1 , wherein the at least one gene comprises at least one gene from Table 6, Table 12, or Table 13. 
     
     
         8 . The method of  claim 1 , wherein the autoimmune disorder is psoriasis. 
     
     
         9 . The method of  claim 1 , wherein the subject has a PASI greater than 8. 
     
     
         10 . The method of  claim 1 , wherein the subject has a PASI of at least 75 following the treating of the subject with the autoimmune therapeutic drug. 
     
     
         11 . The method of  claim 1 , wherein the RNA comprises mRNA or microRNA. 
     
     
         12 . The method of  claim 1 , wherein the collecting a sample comprising RNA derived from skin from the subject comprises penetrating the skin of the subject with a microneedle device, wherein the microneedle device comprises microneedles conjugated to nucleic acid probes. 
     
     
         13 . The method of  claim 1 , further comprising converting the RNA into cDNA and performing next generation sequencing of the cDNA. 
     
     
         14 . The method of  claim 1 , further comprising applying a trained algorithm to data generated by the next generation sequence of the cDNA. 
     
     
         15 . The method of  claim 1 , wherein the at least one gene comprises at least two genes that do not share a common upstream regulator. 
     
     
         16 . The method of  claim 1 , wherein the algorithm was trained using samples from patients that were administered a single type of drug selected from the group consisting of: IL-17 mediated treatment, TNF-alpha mediated treatment and IL-23 mediated treatment. 
     
     
         17 . The method of  claim 1 , wherein the autoimmune therapeutic drug is an IL-17-mediated treatment and the at least one gene comprises at least one gene that is not involved in an IL-17 mediated pathway. 
     
     
         18 . The method of  claim 1 , wherein the autoimmune therapeutic drug is an IL-23-mediated treatment and the at least one gene comprises at least one gene that is not involved in an IL-23 mediated pathway. 
     
     
         19 . The method of  claim 1 , wherein the autoimmune therapeutic drug is a TNF-α-mediated treatment and the at least one gene comprises at least one gene that is not involved in a TNF-α-mediated pathway. 
     
     
         20 . A method of determining whether a skin lesion of a subject will be responsive to an autoimmune therapeutic drug comprising:
 a. penetrating skin of the subject with a microneedle device, wherein the microneedle device comprises one or more nucleic acid probes coupled to a microneedle;   b. removing the microneedle device from the skin of the subject, thereby obtaining RNA molecules from the subject;   c. performing high throughput sequencing on the RNA molecules to generate sequence reads;   d. aligning the sequence reads with a signature of sequence reads associated with a positive response to an autoimmune therapeutic drug to obtain aligned sequence reads; and   e. applying a trained algorithm to the aligned sequence reads, wherein the trained algorithm has a greater than 80% positive predictive value for predicting response to the autoimmune therapeutic drug.   
     
     
         21 . A method of determining whether a skin lesion of a subject will be responsive to an autoimmune therapeutic drug comprising:
 a. penetrating skin of the subject with a microneedle device, wherein the microneedle device comprises one or more nucleic acid probes coupled to a microneedle;   b. removing the microneedle device from the skin of the subject, thereby obtaining RNA molecules from the subject;   c. performing high throughput sequencing on the RNA molecules to generate sequence reads;   d. aligning the sequence reads with a signature of sequence reads associated with a positive response to an autoimmune therapeutic drug to obtain aligned sequence reads;   e. using the aligned sequence reads to determine an expression level of at least one RNA molecule; and   f applying a trained algorithm to the expression level of the at least one RNA molecule, wherein the trained algorithm predicts whether the subject with the skin lesion will be responsive to an IL-17 mediated treatment, an IL-23 mediated treatment, a TNFα mediated treatment, or any combination thereof.   
     
     
         22 . The method of  claim 21 , wherein the trained algorithm predicts whether the subject with the skin lesion will be responsive to the IL-17 mediated treatment. 
     
     
         23 . The method of  claim 21 , wherein the expression level of the at least one RNA molecule corresponds to expression of at least one gene from Table 6, Table 12, or Table 13. 
     
     
         24 . The method of  claim 21 , wherein (b) further comprises:
 i) performing high throughput sequencing on the RNA biomarkers to generate one or more sequence reads for the subject;   ii) aligning the one or more sequence reads for the subject with a known signature of sequence reads, wherein the known signature of sequence reads is associated with a positive response to the recommended treatment, thereby obtaining aligned sequence reads; and   iii) classifying the subject as having a likelihood of positively responding to the recommended treatment by applying a trained algorithm to the aligned sequence reads, wherein the trained algorithm comprises a greater than 50% positive predictive value for predicting a positive response to the recommended treatment.   
     
     
         25 . The method of  claim 25 , wherein the trained algorithm has greater than 50% negative predictive value. 
     
     
         26 . The method of  claim 25 , wherein the recommended treatment comprises one or more autoimmune therapeutic drugs for an autoimmune disease or condition. 
     
     
         27 . The method of  claim 27 , wherein the autoimmune disease or condition is psoriasis. 
     
     
         28 . The method of  claim 25 , wherein the RNA biomarkers are transcribed from at least one gene from Table 6, Table 12 or Table 13. 
     
     
         29 . The method of  claim 25 , wherein the recommended treatment comprises etanercept, infliximab, adalimumab, certolizumab, ustekinumab, secukinumab, ixekizumab, brodalumab, guselkumab, tildrakizumab, risankizumab, or any combination thereof. 
     
     
         30 . A microneedle device comprising:
 a. a microneedle region comprising (i) a microneedle base substrate comprising a first base substrate surface and a second base substrate surface, wherein the first base substrate surface and the second base substrate surface are positioned on opposite sides of the microneedle base substrate; and (ii) a plurality of microneedles protruding from the first base substrate surface; and   b. a support substrate adjacent to the microneedle base substrate, the support substrate connected to or integral with the microneedle base substrate and comprising a support substrate depth, wherein the support substrate depth is greater than the minimum distance between the first base substrate surface and the second base substrate surface.