Methods and compositions for amplification of mRNA

Abstract

To provide methods and compositions for amplification of MRNA.SOLUTION: Provided herein are compositions and methods for the synthesis, amplification and in vitro transcription of full-length cDNA, or cDNA fragments. Methods are provided for reverse transcription of RNA and amplification for in vitro transcription. Further provided are method for loading of dendritic cells with the RNA and homologous lysate for immune stimulation. In a first embodiment, there is provided an oligonucleotide comprising the sequence of SEQ ID NO: 1. In some aspects, the oligonucleotide is further defined as a primer.SELECTED DRAWING: Figure 12

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 599,472, filed December 15, 2017, the entirety of which is incorporated herein by reference.

[0002] Incorporating sequence tables The Sequence Listing, which is 1 KB (measured in Microsoft Windows®), contained in file name “BACMP0006WO__ST25.txt”, created on December 11, 2018, is filed electronically herewith and is hereby incorporated by reference.

[0003] The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to the reverse transcription of mRNA and the amplification of their products. [Background technology]

[0004] An important tool in molecular biology is the ability to replicate DNA and RNA. DNA can be replicated and amplified in vitro by the polymerase chain reaction, where primers hybridize to opposite ends and opposite strands of a target nucleic acid, and the primers are extended by a DNA-dependent DNA polymerase. RNA, which generally exists transiently in vivo, can be reverse transcribed by an RNA-dependent DNA polymerase (reverse transcriptase) to generate a DNA copy that is much more stable than RNA. The DNA produced from reverse transcription, called complementary DNA or cDNA, can be amplified, sequenced, transcribed into RNA, incorporated into a vector for cloning, or a combination of these. There are currently a variety of methods and enzymes for reverse transcribing RNA into DNA. Often, when designing primers for cDNA synthesis from an mRNA template, researchers take advantage of the poly(A) tail present at the 3' end of the mRNA. This allows researchers to use primers with a poly(T) sequence at the 3' end of the primer, greatly simplifying the acquisition of 3' sequence information from the mRNA. The second primer, which corresponds to the 5' end of the target sequence, contains either a random sequence or a target sequence, as in standard PCR. A third option for the 5' primer utilizes the inherent template switching activity of some reverse transcriptases to hybridize the second primer to the polynucleotide overhang generated by the reverse transcriptase and then synthesize a strand complementary to the second primer (see, for example, U.S. Patent No. 5,962,271). The complementary DNA strand can then be copied using a standard PCR reaction to amplify the sequence. Primers used in cDNA synthesis and amplification can include a promoter, which allows the cDNA to be transcribed using an in vitro transcription process to generate large amounts of the target mRNA. RNA generated by in vitro transcription can be used to induce antigen presentation in dendritic cells. However, reverse transcription and in vitro transcription methods suffer from high bias during the reverse transcription process and inefficient in vitro transcription, demonstrating the need for improved methods and compositions to enhance these processes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Pat. No. 5,962,271 Summary of the Invention

[0006] In a first embodiment, an oligonucleotide is provided that comprises the sequence of SEQ ID NO:1. In some aspects, the oligonucleotide is further defined as a primer. In certain aspects, the oligonucleotide comprises less than 40 nucleotides. In certain aspects, the oligonucleotide consists of SEQ ID NO:1.

[0007] In further embodiments, a composition is provided comprising a first primer and a second primer, wherein the first primer comprises the nucleic acid sequence of SEQ ID NO: 1 and the second primer comprises the nucleic acid sequence of SEQ ID NO: 2. In some aspects, the composition is further defined as a nucleic acid amplification reaction mixture. In other aspects, the composition further comprises a third primer, wherein the third primer comprises the nucleic acid sequence of SEQ ID NO: 3.

[0008] In yet a further embodiment, a method is provided for synthesizing a first strand of cDNA from a template mRNA, comprising the steps of: a) hybridizing a first primer to a template mRNA, wherein the first primer comprises the nucleic acid sequence of SEQ ID NO:2; b) extending the first primer using a reverse transcriptase having terminal transferase and template switching activity to generate a partial first strand of cDNA with an oligo(C) overhang; c) hybridizing a second primer to the oligo(C) overhang of the partial first cDNA strand, wherein the second primer comprises the nucleic acid sequence of SEQ ID NO:1; and d) extending the partial first strand of cDNA from the oligo(C) overhang using the second primer as a template, thereby generating a first cDNA strand. In some embodiments, the method further comprises the steps of e) separating the template mRNA and the first cDNA strand; f) hybridizing a third primer to the first cDNA strand; and g) extending the third primer to generate a second cDNA strand, thereby generating a double-stranded cDNA. In certain embodiments, the second primer comprises the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the third primer comprises the nucleic acid sequence of SEQ ID NO: 3. In further embodiments, the template mRNA is obtained from a sample. In some particular embodiments, the sample is a tumor sample. In certain embodiments, the tumor sample is stored in an RNA stabilizing solution after removal from the subject. In still further embodiments, the RNA stabilizing solution is RNALATER®.

[0009] In additional aspects, the method further comprises synthesizing RNA from the double-stranded cDNA. In some aspects, synthesizing RNA from the double-stranded cDNA comprises in vitro transcription. In more specific aspects, the in vitro transcription comprises adding a fourth primer, a fifth primer, and an RNA polymerase, and synthesizing RNA from the double-stranded cDNA with the RNA polymerase. In a particular aspect, the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 1. In a further aspect, the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 3. In another aspect, the method further comprises capping the RNA.

[0010] In certain embodiments, the method further comprises amplifying the double-stranded cDNA. In some embodiments, the second primer comprises the nucleic acid sequence of SEQ ID NO: 1. In other embodiments, the third primer comprises the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, amplifying the double-stranded cDNA comprises adding a DNA-dependent DNA polymerase, a fourth primer, and a fifth primer, and amplifying the cDNA by polymerase chain reaction. In some particular embodiments, the fourth primer comprises the nucleic acid sequence of SEQ ID NO: 1. In another embodiment, the fifth primer comprises the nucleic acid sequence of SEQ ID NO: 3.

[0011] In further embodiments, the method further comprises in vitro transcribing the amplified cDNA to produce a sense strand amplified mRNA. In some embodiments, the second primer comprises the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the third primer comprises the nucleic acid sequence of SEQ ID NO: 3. In some further embodiments, the in vitro transcription of the amplified cDNA comprises adding primers having the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 to the amplified cDNA and hybridizing the primers to the amplified cDNA. In certain embodiments, the in vitro transcription of the amplified cDNA comprises adding an RNA polymerase to the amplified cDNA and extending the hybridized primers to produce RNA. In another embodiment, the method further comprises capping the amplified RNA.

[0012] Further embodiments of the invention provide methods of transducing a dendritic cell population, the methods comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, the nucleic acid comprising RNA produced by any of the methods and aspects described above. In some aspects, the methods further comprise contacting the dendritic cell population with a tumor cell lysate. In another aspect, the tumor cell lysate comprises a tumor antigen having an epitope having a sequence that overlaps with a sequence of the nucleic acid encoding the one or more antigens by a minimum of five amino acids.

[0013] In yet another embodiment, a method for providing an immune response to a subject having a diseased cell population is provided, the method comprising: a) obtaining an antigen-stimulated dendritic cell population produced by the method of claim 36 or 37; and b) administering an effective amount of the antigen-stimulated dendritic cell population to the subject. In certain aspects, the antigen-stimulated dendritic cells are stimulated with an antigen associated with cancer, an autoimmune disease, or an infectious disease. In some aspects, the antigen-stimulated dendritic cells are stimulated with at least one tumor antigen. In additional aspects, the method further comprises administering an immune checkpoint inhibitor. In another aspect, the immune checkpoint inhibitor is a CTLA-4 antagonist. In certain aspects, the immune checkpoint inhibitor is ipilimumab, pembrolizumab, or nivolumab.

[0014] Yet further embodiments of the present invention provide a kit comprising the oligonucleotide primer of claim 1, the oligonucleotide primer of SEQ ID NO: 2 and / or the oligonucleotide primer of SEQ ID NO: 3. In some aspects, the kit may further comprise a nucleic acid isolated from a cancer cell, a DNA polymerase, and / or an RNA stabilizing solution. In certain aspects, the RNA stabilizing solution is RNALATER®. In another aspect, the kit is further defined as a cDNA synthesis kit. In additional aspects, the kit further comprises dNTPs, MgCl2, reverse transcriptase, and / or RNase inhibitor.

[0015] As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the particular component resulting from any unintentional contamination of the composition is preferably less than 0.01%. Most preferred are compositions in which the amount of any of the particular components cannot be detected by standard analytical methods.

[0016] As used in the specification and claims, "a" or "an" may mean one or more. When used in conjunction with the term "comprising," the words "a" or "an" as used in the specification and claims may mean one or more. As used in the specification and claims, "another" or "further" may mean at least a second or more.

[0017] As used in this specification and claims, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists among study subjects.

[0018] Other objects, features, and advantages of the present invention will become apparent from the following detailed description, but this detailed description and specific examples are given by way of illustration only, while illustrating specific embodiments of the present invention, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. In certain embodiments, for example, the following are provided: (Item 1) An oligonucleotide comprising the sequence of SEQ ID NO:1. (Item 2) 2. The oligonucleotide of claim 1, wherein the oligonucleotide is further defined as a primer. (Item 3) 2. The oligonucleotide of claim 1, wherein the oligonucleotide comprises less than 40 nucleotides. (Item 4) 2. The oligonucleotide according to item 1, wherein the oligonucleotide consists of SEQ ID NO:1. (Item 5) A composition comprising a first primer and a second primer, wherein the first primer comprises the nucleic acid sequence of SEQ ID NO:1 and the second primer comprises the nucleic acid sequence of SEQ ID NO:2. , the composition. (Item 6) 6. The composition of claim 5, wherein the composition is further defined as a nucleic acid amplification reaction mixture. (Item 7) 7. The composition of claim 5 or 6, further comprising a third primer, wherein the third primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 8) 8. The composition of claim 7, further comprising a nucleic acid. (Item 9) 9. The composition of claim 8, wherein the nucleic acid is RNA. (Item 10) A method for synthesizing a first strand cDNA from a template mRNA, comprising: a) hybridizing a first primer to the template mRNA, wherein the first primer comprises the nucleic acid sequence of SEQ ID NO:2; b) extending the first primer using a reverse transcriptase having terminal transferase and template switching activity to generate a partial first strand cDNA with an oligo(C) overhang; c) hybridizing a second primer to the oligo(C) overhang of the partial first strand cDNA, wherein the second primer comprises the nucleic acid sequence of SEQ ID NO:1; d) extending the partial first strand cDNA from the oligo(C) overhang using the second primer as the template, thereby generating the first strand cDNA. (Item 11) e) separating the template mRNA and the first strand cDNA; f) hybridizing a third primer to the first strand cDNA; g) extending the third primer to generate a second strand of cDNA, thereby generating a double-stranded cDNA. (Item 12) 12. The method of claim 11, wherein the second primer comprises the nucleic acid sequence of SEQ ID NO:1. (Item 13) 12. The method of claim 11, wherein the third primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 14) 12. The method of claim 11, wherein the template mRNA is obtained from a sample. (Item 15) 15. The method of claim 14, wherein the cell sample is a tumor sample. (Item 16) 16. The method of claim 15, wherein the tumor sample is stored in an RNA stabilizing solution after removal from the subject. (Item 17) 17. The method of claim 16, wherein the RNA stabilizing solution is RNALATER®. (Item 18) 12. The method of claim 11, further comprising synthesizing RNA from the double-stranded cDNA. (Item 19) 20. The method of claim 18, wherein said synthesizing RNA from said double-stranded cDNA comprises in vitro transcription. (Item 20) In vitro transcription includes adding a fourth primer, a fifth primer, and an RNA polymerase, and using the RNA polymerase to transcribe a cDNA fragment from the double-stranded cDNA. 20. The method according to item 19, comprising synthesizing RNA from the nucleic acid sequence. (Item 21) 21. The method of claim 20, wherein the fourth primer comprises the nucleic acid sequence of SEQ ID NO:1. (Item 22) 22. The method of claim 21, wherein the fourth primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 23) 21. The method of claim 20, further comprising capping the RNA. (Item 24) 12. The method of claim 11, further comprising amplifying the double-stranded cDNA. (Item 25) 25. The method of claim 24, wherein the second primer comprises the nucleic acid sequence of SEQ ID NO:1. (Item 26) 25. The method of claim 24, wherein the third primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 27) 25. The method of claim 24, wherein amplifying the double-stranded cDNA comprises adding a DNA-dependent DNA polymerase, a fourth primer, and a fifth primer, and amplifying the cDNA by polymerase chain reaction. (Item 28) 28. The method of claim 27, wherein the fourth primer comprises the nucleic acid sequence of SEQ ID NO:1. (Item 29) 28. The method of claim 27, wherein the fifth primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 30) 25. The method of claim 24, further comprising in vitro transcribing the amplified cDNA to produce a sense strand amplified mRNA. (Item 31) 31. The method of claim 30, wherein the second primer comprises the nucleic acid sequence of SEQ ID NO:1. (Item 32) 31. The method of claim 30, wherein the third primer comprises the nucleic acid sequence of SEQ ID NO:3. (Item 33) 31. The method of claim 30, wherein the in vitro transcription of the amplified cDNA comprises adding primers having the sequences of SEQ ID NO:1 and SEQ ID NO:3 to the amplified cDNA and hybridizing the primers to the amplified cDNA. (Item 34) 34. The method of claim 33, wherein the in vitro transcription of the amplified cDNA comprises adding an RNA polymerase to the amplified cDNA and extending the hybridized primer to produce RNA. (Item 35) 31. The method of claim 30, further comprising capping the amplified RNA. (Item 36) 36. A method for transducing a dendritic cell population, the method comprising contacting the dendritic cell population with a nucleic acid encoding one or more antigens, the nucleic acid comprising RNA produced by the method of any one of items 18 to 35. (Item 37) 37. The method of claim 36, further comprising contacting the population of dendritic cells with a tumor cell lysate. (Item 38) 38. The method of claim 37, wherein the tumor cell lysate comprises a tumor antigen having an epitope having a sequence that overlaps by at least five amino acids with a sequence of the nucleic acid encoding one or more antigens. (Item 39) 1. A method for providing an immune response to a subject having a diseased cell population, comprising: a) obtaining a population of antigen-stimulated dendritic cells produced by the method according to item 36 or 37; b) administering to the subject an effective amount of the antigen-stimulated dendritic cell population. (Item 40) 40. The method of claim 39, wherein the antigen-stimulated dendritic cells are stimulated with an antigen associated with cancer, an autoimmune disease, or an infectious disease. (Item 41) The method of claim 40, wherein the antigen-stimulated dendritic cells are stimulated with at least one tumor antigen. (Item 42) 42. The method of item 41, further comprising administering an immune checkpoint inhibitor. (Item 43) 43. The method of claim 42, wherein the immune checkpoint inhibitor is a CTLA-4 antagonist. (Item 44) 43. The method of item 42, wherein the immune checkpoint inhibitor is ipilimumab, pembrolizumab or nivolumab. (Item 45) A kit comprising the oligonucleotide primer according to item 1, the oligonucleotide primer of SEQ ID NO: 2 and / or the oligonucleotide primer of SEQ ID NO: 3. (Item 46) 46. ​​The kit of item 45, further comprising nucleic acid isolated from a cancer cell. (Item 47) 46. ​​The kit according to item 45, further comprising a DNA polymerase. (Item 48) 46. ​​The kit of item 45, further comprising an RNA stabilization solution. (Item 49) 49. The kit of item 48, wherein the RNA stabilizing solution is RNALATER®. (Item 50) 46. ​​The kit of claim 45, wherein the kit is further defined as a cDNA synthesis kit. (Item 51) 51. The kit of item 50, further comprising dNTPs, MgCl2, reverse transcriptase, and / or an RNase inhibitor.

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0020] [Figure 1]Freshly harvested tumor samples produce higher quality RNA than cryopreserved samples. Photographs of RNA isolated from either frozen or fresh tumor tissue run on a non-denaturing 1% agarose gel. From left to right: L: ladder. 1: frozen tumor sample RNA; 2 frozen tumor sample RNA; 3: frozen tumor sample RNA; 4: fresh tumor sample RNA. [Diagram 2] Comparison of two different RNA isolation methods. Photographed RNA samples were run on a non-denaturing 1% agarose gel. Lane 1: RNA isolated from 30 mg of tumor tissue by column purification. Lane 2: RNA isolated from 30 mg of tumor tissue by guanidinium thiocyanate / phenol / chloroform purification. [Diagram 3] 1 is a schematic diagram showing the process of reverse transcription used in the present invention, showing the primers used: VKWD primer has the nucleotide sequence of SEQ ID NO: 1; CDS64T+ oligo primer has the nucleotide sequence of SEQ ID NO: 2; T7 power switch primer has the nucleotide sequence of SEQ ID NO: 3. [Figure 4] FIG. 1 shows a comparison of methods for reverse transcription and subsequent in vitro transcription. In the picture, four different protocols were used to generate in vitro transcribed RNA from the same starting material. Lane 1, uncapped transcript generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Lane 2, capped transcript generated by the Slagter-Jager method (Slagter-Jager et al., 2013). Lane 3, uncapped RNA generated by the methods described herein. Lane 4, capped RNA generated by the methods described herein. [Diagram 5]Comparison of capped mRNA produced by two different methods. 5ug of in vitro transcribed mRNA was run on a 1% agarose non-denaturing gel. Lane 1 shows mRNA produced by the previous method (Slagter-Jager et al., 2013) that has a post-transcriptional cap structure. Lane 2 shows mRNA produced by the current method that is co-transcriptionally capped. [Figure 6] IFNγ production is significantly enhanced by T cells co-cultured with dendritic cells loaded with capped mRNA and lysate. Photographs show the relative amounts of IFN-γ secretion measured by ELISA for cells loaded with uncapped or capped mRNA and lysate produced by previous (Argos (Slagter-Jager)) or current (VKWD) methods, or with mismatched mRNA and lysate. [Figure 7] Figure 1 shows that production of antigen-specific CD8+CD25+ T cells is increased when co-cultured with dendritic cells homologously loaded with capped mRNA and lysates. The photograph shows flow cytometric analysis of T cells for antigen-specific activation markers after restimulation with mRNA-loaded dendritic cells, as indicated below the data. [Figure 8] Figure 1 shows a Western blot analysis of dendritic cell lysates. Lane 1: unloaded dendritic cells. Lane 2: dendritic cells loaded with uncapped mRNA generated using the Slagter-Jager method. Lane 3: dendritic cells loaded with capped mRNA generated using the Slagter-Jager method. Lane 4: dendritic cells loaded with uncapped mRNA generated using the methods described herein. Lane 5: dendritic cells loaded with capped mRNA generated using the methods described herein. Lane 6: dendritic cells loaded with mismatched mRNA and lysate. Lane 7: dendritic cells loaded with co-transcriptionally capped mRNA generated using the methods described herein. [Figure 9] Figure 1 shows that homologous antigen loading with in vitro transcribed and amplified mRNA increases retention of CTLA4. Picture shows flow cytometric analysis of dendritic cells loaded with unloaded or indicated mRNA, lysate, 2x (mRNA and lysate), and MM (mismatched mRNA and lysate). [Figure 10] Figure 1 shows enhanced retention of CTLA4 and decreased release of CTLA4 from homologously loaded dendritic cells. Photograph shows a Western blot detecting CTLA4 in supernatants or lysates of dendritic cell cultures. Dendritic cells were either unloaded (UL) or loaded with mRNA, lysate, both, or mismatched mRNA and lysate. mRNA was isolated by either a column-based method or the guanidinium thiocyanate / phenol / chloroform method. [Figure 11] Figure 1 shows that IL-12 transcript levels are elevated in dendritic cells homologously loaded with lysates and in vitro transcribed mRNA. IL-12a and IL-12b levels detected by ELISA are shown for dendritic cells loaded with the indicated RNAs and / or lysates. The left axis shows arbitrary transcription units normalized to unloaded control DCs and set to 1.0. [Figure 12] FIG. 1 shows a flow chart illustrating the preparation of a dendritic cell vaccine. [Figure 13A] Amplified mRNA generates TH1 immune responses in vitro as efficiently as native polyA mRNA when loaded into DCs with homologous lysates. Monocyte-derived human DCs were loaded with a) native polyA tumor mRNA and homologous / heterologous lysates or 2) capped IVT amplified mRNA and homologous / heterologous lysates. DCs were then co-cultured with T cells and the percentage of activated CD8+CD25+Ifng+ cells was analyzed by flow cytometry. T cells co-cultured with unloaded DCs or with uncapped mRNA-loaded DCs served as controls. [Figure 13B] FIG. 1 shows that amplified mRNA generates TH1 immune responses in vitro as efficiently as native polyA mRNA when co-loaded with homologous lysates into DCs. DCs loaded with various combinations of polyA mRNA or IVT amplified mRNA and homologous or heterologous cell lysates were matured for 48 h and intracellular CTLA4 levels were analyzed by flow cytometry. Singly loaded and unloaded DCs serve as controls. Upregulation of intracellular CTLA-4 indicates increased retention and therefore decreased secretion. UL-non-loaded DCs, uncapped mRNA-DCs loaded with uncapped IVT mRNA, mRNA-polyA mRNA-loaded DCs, lysate-lysate-loaded DCs, 2xw / uncapped-DCs loaded with uncapped IVT mRNA and homologous lysate, 2x capped-DCs loaded with capped IVT mRNA and homologous lysate, uncapped MM-DCs loaded with uncapped IVT mRNA and heterologous lysate, capped MM-DCs loaded with capped IVT mRNA and heterologous lysate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Reverse transcription, a process in which RNA-dependent DNA polymerase synthesizes DNA using RNA as a template, has become an increasingly important tool in molecular biology for understanding gene expression and sequencing cellular RNA. Using the process of in vitro transcription and amplification followed by reverse transcription, large amounts of RNA can be generated from a template RNA. However, methods are needed to reduce bias and increase the efficiency of the reverse transcription process.

[0022] Therefore, in certain embodiments, the present disclosure provides a method for reducing the bias of the reverse transcription process and increasing the amplification efficiency of reverse transcribed molecules.In particular, the present study found that for efficient in vitro transcription, the nucleotide composition and sequence of the primer are important for the reverse transcription process and the incorporation of a promoter sequence in the primer.

[0023] Specifically, in some embodiments, compositions and methods are provided for the synthesis, amplification, and in vitro transcription of full-length cDNA or cDNA fragments. The methods may include contacting RNA with a primer capable of annealing to the poly(A) tail of an mRNA, a suitable enzyme with reverse transcriptase activity, and a template-switching oligonucleotide under conditions sufficient to allow template-dependent extension of the primer to a cDNA complementary to the mRNA template. The template-switching oligonucleotide hybridizes to the overhang generated by the reverse transcriptase at the 3' end of the newly generated cDNA, allowing the reverse transcriptase to continue synthesizing the complement of the template-switching oligonucleotide at the 3' end of the cDNA. Subsequent amplification can introduce a promoter sequence for in vitro transcription. The in vitro transcribed RNA can then be loaded into dendritic cells along with a homologous lysate for immune stimulation. Thus, in further embodiments, methods for loading dendritic cells and the use of dendritic cell vaccines for the treatment of diseases such as cancer are provided.

[0024] definition "Amplification," as used herein, refers to an in vitro process for increasing the number of copies of a nucleotide sequence(s). Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 cycles of denaturation and replication.

[0025] "Polymerase chain reaction" or "PCR" refers to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicas of a target nucleic acid flanked by primer binding sites, such a reaction comprising the repetition of one or more of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers with a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler machine. The specific temperatures, durations at each step, and rates of change between steps depend on many factors well known to those skilled in the art and are described, for example, in reference: McPherson et al., editors, PCR: A Practical This is exemplified by the book PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995).

[0026] "Primer" refers to an oligonucleotide, either natural or synthetic, that when it forms a duplex with a polynucleotide template, acts as an initiation point for nucleic acid synthesis and can be extended from its 3' end along the template, thereby forming an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Typically, the primer is extended by a DNA polymerase. Primers are generally of a length compatible with their use in synthesizing primer extension products, and typically range from 8 to 100 nucleotides in length, e.g., 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, etc., more typically in the range of 18 to 40, 20 to 35, 21 to 30 nucleotides in length, and any length between the recited ranges. Exemplary primers can range from 10-50 nucleotides in length, e.g., 15-45, 18-40, 20-30, 21-25, etc., and any length between the specified ranges. In some embodiments, primers are typically no more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0027] As used herein, "incorporate" means become a part of a nucleic acid polymer.

[0028] The term "without exogenous manipulation" as used herein refers to the modification of nucleic acid molecules occurring without changing the solution in which the nucleic acid molecules are modified.In certain embodiments, the modification occurs without human hands or machines that change the solution conditions, which may also be called buffer conditions.However, temperature changes may occur during the modification.

[0029] A "nucleoside" is a base-sugar combination, i.e., a nucleotide without a phosphate. It is recognized in the art that there is some interchangeability in the use of the terms nucleoside and nucleotide. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, dUTP functions as a DNA monomer and formally becomes deoxyuridylic acid, i.e., dUMP or deoxyuridine monophosphate. One can say that dUTP is incorporated into DNA even if there is no dUTP moiety in the resulting DNA. Similarly, one can say that deoxyuridine is incorporated into DNA even if it is only part of the substrate molecule.

[0030] As used herein, "nucleotide" is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e., DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.

[0031] The term "nucleic acid" or "polynucleotide" generally refers to at least one molecule or strand of DNA, RNA, DNA-RNA chimera, or derivatives or analogs thereof, that includes at least one nucleic acid base, such as a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A", guanine "G", thymine "T" and cytosine "C") or RNA (e.g., "A", "G", uracil "U" and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide". As used herein, "oligonucleotide" refers collectively and interchangeably to the two technical terms "oligonucleotide" and "polynucleotide". It should be noted that although oligonucleotide and polynucleotide are different technical terms, there is no precise dividing line between them, and they are used interchangeably herein. The term "adapter" may be used interchangeably with the terms "oligonucleotide" and "polynucleotide". Additionally, the term "adapter" may refer to a linear adapter (either single-stranded or double-stranded) or a stem-loop adapter. These definitions generally refer to at least one single-stranded molecule, but in certain embodiments also encompass at least one additional strand that is partially, substantially, or completely complementary to at least one single-stranded molecule. Thus, a nucleic acid may comprise at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule. As used herein, single-stranded nucleic acids are denoted by the prefix "ss" and double-stranded nucleic acids are denoted by the prefix "ds".

[0032] As used herein, "oligonucleotide" refers collectively and interchangeably to the two technical terms "oligonucleotide" and "polynucleotide." It should be noted that although oligonucleotide and polynucleotide are different technical terms, there is no precise boundary between them, and they are used interchangeably herein. The term "adapter" may be used interchangeably with the terms "oligonucleotide" and "polynucleotide."

[0033] "Nucleic acid molecule" or "nucleic acid target molecule" refers to any single-stranded or double-stranded nucleic acid molecule, including standard reference bases, hypermodified bases, non-natural bases, or any combination of those bases. For example, but not limited to, a nucleic acid molecule includes the four reference DNA bases-adenine, cytosine, guanine, and thymine, and / or the four reference RNA bases-adenine, cytosine, guanine, and uracil. If the nucleoside contains a 2'-deoxyribose group, uracil can be used in place of thymine. Nucleic acid molecules can be converted from RNA to DNA and from DNA to RNA. For example, but not limited to, mRNA can be made into complementary DNA (cDNA) using reverse transcriptase, and DNA can be made into RNA using RNA polymerase. Nucleic acid molecules can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, DNA / RNA hybrids, amplified DNA, existing nucleic acid libraries, and the like. Nucleic acids can be obtained from human samples such as blood, serum, plasma, cerebrospinal fluid, cheek scraping, biopsy, semen, urine, feces, saliva, sweat, etc. Nucleic acid molecules may be subjected to various treatments, such as repair and fragmentation treatments. Fragmentation treatments include mechanical, sonic and hydrodynamic shearing. Repair treatments include repairing nicks by extension and / or ligation, polishing to create blunt ends, and removing damaged bases such as deamination, derivatization, abasic, or crosslinked nucleotides. Nucleic acid molecules of interest may also be subjected to chemical modification (e.g., bisulfite conversion, methylation / demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.

[0034] "Analog" forms of purines and pyrimidines are well known in the art and include, but are not limited to, aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid methyl ester, pseudouracil, quasine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine. Nucleic acid molecules may also contain one or more hypermodified bases, such as, but not limited to, 5-hydroxymethyluracil, 5-hydroxyuracil, a-putresinylthymine, 5-hydroxymethylcytosine, 5-hydroxycytosine, 5-methylcytosine, N 4 -Methylcytosine, 2-aminoadenine, a-carbamoylmethyladenine, N 6 -Methyladenine, inosine, xanthine, hypoxanthine, 2,6-diaminpurine, and N 7-methylguanine. The nucleic acid molecule may also include one or more unnatural bases, such as, but not limited to, 7-deaza-7-hydroxymethyladenine, 7-deaza-7-hydroxymethylguanine, isocytosine (isoC), 5-methylisocytosine, and isoguanine (isoG). Nucleic acid molecules containing only standard, hypermodified, unnatural bases, or any combination of such bases, can, for example and without limitation, each linkage between nucleotide residues can consist of a standard phosphodiester linkage, and may further include substitution of a non-bridging oxygen atom with a nitrogen atom (i.e., a phosphoramidate linkage), a sulfur atom (i.e., a phosphorothioate linkage), or an alkyl or aryl group (i.e., an alkyl or aryl phosphonate), substitution of a bridging oxygen atom with a sulfur atom (i.e., a phosphorothioate), substitution of a phosphodiester bond with a peptide bond (i.e., a peptide nucleic acid or PNA), or formation of one or more additional covalent bonds (i.e., a locked nucleic acid or LNA), which has an additional bond between the 2'-oxygen and 4'-carbon of the ribose sugar.

[0035] A nucleic acid that is "complementary" or "complement(s)" is one that can base pair according to standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein, the term "complementary" or "complement(s)" can refer to a nucleic acid that is substantially complementary, as can be assessed by the same nucleotide comparison above. The term "substantially complementary" refers to a nucleic acid that comprises at least one sequence of contiguous nucleobases, or, if one or more nucleobase portions are not present in the molecule, semi-contiguous nucleobases can hybridize to at least one nucleic acid strand or duplex, even if not all nucleobases are base paired with the corresponding nucleobase. In certain embodiments, a "substantially complementary" nucleic acid comprises at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% to about 100%, and any range therein, of the nucleobase sequence can form base pairs with at least one single-stranded or double-stranded nucleic acid molecule during hybridization. In certain embodiments, the term "substantially complementary" refers to at least one nucleic acid that can hybridize to at least one nucleic acid strand or duplex under stringent conditions. In certain embodiments, a "partially complementary" nucleic acid comprises at least one sequence that can hybridize to at least one single-stranded or double-stranded nucleic acid under low stringency conditions, or comprises at least one sequence in which less than about 70% of the nucleic acid base sequence can form base pairs with at least one single-stranded or double-stranded nucleic acid molecule during hybridization.

[0036] The term "non-complementary" refers to a nucleic acid sequence that does not have the ability to form at least one Watson-Crick base pair through specific hydrogen bonds.

[0037] As used herein, the term "blunt end" refers to an end of a dsDNA molecule having a 5' end and a 3' end, where the 5' end and the 3' end terminate at the same nucleotide position. Thus, a blunt end does not include a 5' or 3' overhang.

[0038] As used herein, the term "overhang" refers to the end of a dsDNA molecule having a 5' end and a 3' end, which terminate at different nucleotide positions such that there is at least one nucleotide remaining at either the 5' end or the 3' end that has no nucleotides to hydrogen bond with.

[0039] The term "cap" as used herein refers to a guanine nucleoside that is attached through its 5'-carbon to a triphosphate group and then to the 5'-carbon of most 5'-nucleotides of primary mRNA transcripts, and in most eukaryotes, the nitrogen at the 7th position of the guanine in the capped nucleotide is methylated. Most eukaryotic cellular mRNA transcripts and most eukaryotic viral mRNA transcripts are blocked or "capped" at the 5' end. In addition to mRNAs, other forms of eukaryotic RNA, including but not limited to small nuclear RNAs ("snRNAs") and pre-microRNAs (i.e., "pre-miRNAs", primary transcripts that are processed to become miRNAs), also have cap structures. The 5' caps of eukaryotic cellular and viral mRNAs (and some other forms of RNA) play important roles in RNA stability and processing. For example, caps are required to modify the extent of processing and maturation of RNA transcripts in the nucleus, transport of mRNAs from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNAs into proteins.

[0040] By "sample" is meant a material obtained or isolated from a fresh or preserved biological sample or synthetically produced source that contains a nucleic acid of interest. In certain embodiments, a sample is a biological material that contains the variable immune region(s) for which data or information is sought. Samples can include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tears, vaginal secretions, sweat, lymphatic fluid, cerebrospinal fluid, mucosal secretions, peritoneal fluid, ascites fluid, fecal material, bodily fluids, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing the immune nucleic acid of interest. Samples can also include non-human sources such as non-human primates, rodents and other mammals, other animals, plants, fungi, bacteria, and viruses.

[0041] As used herein with respect to a nucleotide sequence, "substantially known" refers to having sufficient sequence information to allow for preparation of a nucleic acid molecule, such as its amplification. This is typically about 100%, but in some embodiments, some of the adapter sequences are random or degenerate. Thus, in certain embodiments, substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0042] Nucleic Acids of the Embodiments Nucleic acids of the present disclosure may include amplification, reverse transcription, and / or in vitro transcription primers and oligonucleotides, tumor-derived nucleic acids, and / or recombinant nucleic acids encoding one or more proteins or peptides, such as those capable of inducing an immune response that is effective against a tumor or tumor cells. In some embodiments, the generation of nucleic acids capable of inducing an immune response includes reverse transcription of a target mRNA to generate a cDNA. Methods of reverse transcription are well known in the art (U.S. Pat. Nos. 5,962,271 and 6,518,019, which are incorporated herein by reference). Reverse transcription is performed by an RNA-dependent DNA polymerase, also known as reverse transcriptase. Reverse transcriptase binds to a primer hybridized to the 3' end of the target mRNA and synthesizes a DNA complementary to the mRNA. Reverse transcriptase may add some non-templated nucleotides, such as a short poly(C) tail, to the 3' end of the newly synthesized cDNA. A second primer can be hybridized to the 3' end of the nascent cDNA and the reverse transcriptase can continue synthesizing DNA complementary to the primer using template switching activity to use the second primer as its template.

[0043] Primers or oligonucleotides used in reverse transcription or amplification may allow for the addition of exogenous DNA or RNA sequences to the 5' and 3' ends of the target sequence during complementary strand synthesis. Primers or oligonucleotides used to prime the reverse transcription or amplification reaction may incorporate a variety of features, including but not limited to transcription promoters, ribosome binding sites, or restriction endonuclease cleavage sites. In some embodiments, primers may include transcription promoter sequences.

[0044] The cDNA prepared by reverse transcription can be amplified or transcribed into RNA. To increase the concentration of cDNA, the cDNA can be amplified by PCR. The primer for reverse transcription can be used as a primer for PCR, or another primer can be added. The PCR reaction can utilize a reverse transcriptase to amplify the cDNA, or a DNA-dependent DNA polymerase can be added to increase the efficiency of DNA amplification. The primer for reverse transcription or DNA amplification can include a transcription promoter sequence, such as a T7 promoter sequence. The cDNA or amplified cDNA can be transcribed in an in vitro transcription process to generate large amounts of initial target RNA using any commercially available RNA polymerase or in vitro transcription kit, such as AMPLISCRIBE™ T7-FLASH® (LUCIGEN® Catalog No. ASF3257).

[0045] In certain embodiments, the nucleic acid composition comprises both a tumor-derived nucleic acid population and a recombinant nucleic acid component, the combined nucleic acid composition being enriched for known tumor antigens, or enriched for specific or other nucleic acids encoding proteins or peptides that enhance the effectiveness of the methods and compositions described herein.

[0046] "Tumor-derived" nucleic acid refers to nucleic acid that contains RNA whose origin is in a tumor cell and corresponds to a tumor antigen(s). Included is RNA that encodes all or part of a tumor antigen or a previously identified tumor antigen. Such nucleic acid can be "in vitro transcribed". For example, it can be reverse transcribed and amplified by PCR to generate cDNA that can then be transcribed in vitro with or without cloning the cDNA. Also included is RNA provided as a fractionated preparation of tumor cells. Identification of tumor antigens is not required, as even unfractionated RNA preparations (e.g., total RNA or total polyA RNA) can be used. In one embodiment, the preparation is fractionated with respect to non-RNA component(s) of cells. This is to enrich the preparation for RNA by reducing the concentration of non-RNA components such as proteins, lipids and / or DNA. If desired, the preparation is further fractionated with respect to RNA (e.g., by subtractive hybridization) to generate "tumor-specific" or "pathogen-specific" RNA.

[0047] "Tumor-specific" RNA refers to an RNA sample that has a high content of RNA that is preferentially present in tumor cells compared to non-tumor cells, relative to unfractionated tumor-derived RNA. For example, tumor-specific RNA includes RNA that is present in tumor cells but not in non-tumor cells. This definition also includes RNA samples that contain RNA that is present in both tumor and non-tumor cells, but at higher levels in tumor cells than in non-tumor cells. This definition also includes RNA that encodes previously identified tumor antigens and is generated in vitro, for example from a plasmid or by PCR. Alternatively, tumor-specific RNA can be prepared by fractionating a tumor sample such that the proportion of RNA corresponding to the tumor antigen is increased compared to unfractionated tumor-derived RNA. For example, tumor-specific RNA can be prepared by fractionating tumor-derived RNA using conventional subtractive hybridization techniques to RNA from non-tumor cells.

[0048] Methods are provided herein that are suitable for the generation of tumor-derived nucleic acids or RNA. These nucleic acids can be used for antigenic stimulation of dendritic cells and for the preparation of mature dendritic cells. It is not necessary that the nucleic acids be provided to DCs in purified form. Preferably, the RNA sample (i.e., fractionated tumor preparation) is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (wt / vol) RNA.

[0049] Transfection methods known in the art are suitable for introducing tumor-derived nucleic acids into dendritic cells. For example, 5-50 μg of RNA in 500 μl of Opti-MEM can be mixed with cationic lipids at a concentration of 10-100 μg and incubated at room temperature for 20-30 minutes. Other suitable lipids include LIPOFECTIN™ (1:1 (w / w) DOTMA:DOPE), LIPOFECTAMINE™ ((3:1 (w / w) DOSPA:DOPE), DODAC:DOPE (1:1), CHOL:DOPE (1:1), DMEDA, CHOL, DDAB, DMEDA, DODAC, DOPE, DORI, DORIE, DOSPA, DOTAP, and DOTMA. The resulting RNA-lipid complex is then added to 1-3x106 cells, preferably 2x106 antigen presenting cells, and incubated for 2-4 hours at 37°C in a total volume of about 2 ml (e.g., in Opti-MEM). Alternatively, RNA can be introduced into the antigen presenting cells using conventional techniques such as electroporation or calcium phosphate transfection using 1-5x106 cells and 5-50 μg of RNA. Typically, 5-20 μg of polyA RNA or 25-50 µg of total RNA are typically used.

[0050] When RNA is provided as tumor preparation, typically, the preparation is fractionated or otherwise processed to reduce the concentration of protein, lipid, and / or DNA in the preparation, and enrich the preparation of RNA.For example, RNA can be at least partially purified from tumor cells or pathogens using RNA purification methods known in the art.RNA preparation can also be treated with protease or RNase-free DNase.

[0051] Dendritic Cell Populations of the Invention Methods for isolating, culturing and priming dendritic cells are well known in the art. For example, U.S. Patent No. 8,728,806, the entirety of which is incorporated herein by reference, provides detailed methods for providing antigen-primed dendritic cells that can be used in the compositions and methods of the embodiments. In certain aspects, dendritic cells for use according to the embodiments are isolated from a subject to be treated by the methods of the embodiments. In other aspects, the dendritic cells can be derived from a different subject, such as an HLA-matched donor. In certain aspects, the dendritic cells are derived from a bank of dendritic cells with defined HLA typing. In a preferred aspect, the antigen-primed dendritic cells for use according to the embodiments are homologously loaded with antigen, as detailed herein and in U.S. Patent No. 8,728,806.

[0052] Methods for isolating enriched cell populations of dendritic cell precursors and immature dendritic cells from various sources, such as blood and bone marrow, are known in the art. For example, dendritic cell precursors and immature dendritic cells can be isolated by drawing heparinized blood, by apheresis or leukapheresis, by preparation of buffy coats, formation of rosette-like structures, centrifugation, density gradient centrifugation (e.g., using FICOLL (e.g., FICOLL-PAQUE®), PERCOLL® (colloidal silica particles (15-30 mm diameter) coated with non-dialyzable polyvinylpyrrolidone (PVP), sucrose, etc.), differential lysis of cells, filtration, etc. In certain embodiments, leukocyte populations can be prepared, for example, by collecting blood from a subject, triturating to remove platelets, lysing red blood cells, etc. Dendritic cell precursors and immature dendritic cells can optionally be enriched for monocytic dendritic cell precursors, for example, by centrifugation through a PERCOLL® gradient. In other embodiments, dendritic cell precursors can be selected using CD14 selection of G-CSF mobilized peripheral blood.

[0053] Dendritic cell precursors and immature dendritic cells may optionally be prepared in a closed, sterile system. As used herein, the term "closed sterile system" or "closed system" refers to a system in which exposure to non-sterile ambient or circulating air or other non-sterile conditions is minimized or eliminated. A closed system for isolating dendritic cell precursors and immature dendritic cells generally excludes density gradient centrifugation in open-top tubes, open-air transfer of cells, cell culture in tissue culture plates or non-sealed flasks, and the like. In an exemplary embodiment, the closed system allows for the sterile transfer of dendritic cell precursors and immature dendritic cells from their initial collection container to a closed tissue culture container without exposure to non-sterile air.

[0054] In certain embodiments, monocytic dendritic cell precursors are isolated by attachment to a monocyte-binding substrate. For example, a population of leukocytes (e.g., isolated by leukapheresis) can be contacted with a substrate to which monocytic dendritic cell precursors are attached. When the population of leukocytes contacts the substrate, monocytic dendritic cell precursors in the population of leukocytes preferentially adhere to the substrate. Other leukocytes (such as other potential dendritic cell precursors) exhibit reduced binding affinity to the substrate, thereby allowing monocytic dendritic cell precursors to be preferentially enriched on the surface of the substrate.

[0055] Suitable substrates include, for example, those with a large surface area to volume ratio. Such substrates can be, for example, particulate or fibrous substrates. Suitable particulate substrates include, for example, glass particles, plastic particles, glass-coated plastic particles, glass-coated polystyrene particles, and other beads suitable for absorbing proteins. Suitable fibrous substrates include microcapillary and microvillous membranes. Particulate or fibrous substrates are typically capable of eluting attached monocytic dendritic cell precursors without substantially reducing the viability of the attached cells. Particulate or fibrous substrates can be substantially non-porous to facilitate elution of monocytic dendritic cell precursors or dendritic cells from the substrate. A "substantially non-porous" substrate is one in which at least the majority of pores present in the substrate are smaller than the cells, to at least minimize the incorporation of cells in the substrate.

[0056] The attachment of monocytic dendritic cell precursors to the substrate can be optionally increased by adding a binding medium. Suitable binding media can include monocytic dendritic cell precursor medium (e.g., AIM-V®, RPMI1640, DMEM, X-VIVO15®, etc.) supplemented individually or in any combination with cytokines (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), or interleukin 13 (IL-13)), plasma, serum (e.g., human serum, such as autologous or allogeneic serum), purified proteins, such as serum albumin, divalent cations (e.g., calcium and / or magnesium ions), and other molecules that favor specific attachment of monocytic dendritic cell precursors to the substrate or prevent attachment of non-monocytic dendritic cell precursors to the substrate. In certain embodiments, the plasma or serum can be heat inactivated. Heat inactivated plasma can be autologous or xenogeneic to the leukocytes.

[0057] After the monocytic dendritic cell precursors adhere to the substrate, the non-adherent leukocytes are separated from the monocytic dendritic cell precursor / substrate complexes. Any suitable means can be used to separate the non-adherent cells from the complexes. For example, the mixture of non-adherent leukocytes and the complexes can be allowed to settle, and the non-adherent leukocytes and medium can be decanted or discarded. Alternatively, the mixture can be centrifuged, and the supernatant containing the non-adherent leukocytes can be decanted or discarded from the pelleted complexes.

[0058] Isolated dendritic cell precursors may be cultured ex vivo for differentiation, maturation and / or expansion. (As used herein, isolated immature dendritic cells, dendritic cell precursors, T cells, and other cells refer to cells that exist by human hands away from their native environment and thus are not the product of nature. Isolated cells may exist in purified, semi-purified, or non-native environments.) Briefly, ex vivo differentiation typically involves culturing dendritic cell precursors, or a population of cells having dendritic cell precursors, in the presence of one or more differentiation agents. Suitable differentiation agents may be, for example, cell growth factors (e.g., cytokines such as (GM-CSF), interleukin 4 (IL-4), interleukin 13 (IL-13), and / or combinations thereof). In certain embodiments, monocytic dendritic cell precursors are differentiated to form monocyte-derived immature dendritic cells.

[0059] The dendritic cell precursors can be cultured and differentiated under suitable culture conditions. Suitable tissue culture media include AIM-V®, RPMI1640, DMEM, X-VIVO15®, and the like. The tissue culture media can be supplemented with serum, amino acids, vitamins, as well as cytokines such as GM-CSF and / or IL-4, divalent cations, and the like to promote differentiation of the cells. In certain embodiments, the dendritic cell precursors can be cultured in serum-free media. Such culture conditions can optionally exclude any animal-derived products. A typical cytokine combination in a typical dendritic cell culture medium is about 500 units / ml each of GM-CSF (50 ng / ml) and IL-4 (10 ng / ml). When the dendritic cell precursors differentiate to form immature dendritic cells, they are nearly phenotypically similar to Langerhans cells of the skin. Immature dendritic cells are typically CD14- and CD11c+, express low levels of CD86 and CD83, and can capture soluble antigens via specialized endocytosis. Immature DCs expressed very high levels of CD86. The population was also mixed with respect to CD14 and CD11C. The majority were CD11c+, but there were distinct subpopulations that were CD11c- and CD14+.

[0060] Immature dendritic cells mature to form mature dendritic cells. Mature DCs lose the ability to take up antigens and display upregulated expression of costimulatory cell surface molecules and various cytokines. Specifically, mature DCs express higher levels of MHC class I and II antigens than immature dendritic cells, and mature dendritic cells are generally identified as CD80+, CD83+, CD86+, and CD14-. Increased expression of MHC increases antigen density on the DC surface, while upregulation of costimulatory molecules CD80 and CD86 enhances T cell activation signals through their counterparts, such as CD28 on T cells.

[0061] The mature dendritic cells of the present invention can be prepared (i.e., matured) by contacting immature dendritic cells with an effective amount or concentration of a nucleic acid composition and a tumor antigen composition. An effective amount of the nucleic acid composition typically ranges from up to, at least, or about 0.01, 0.1, 1, 5, 10 to 10, 15, 20, 50, 100 ng or mg of nucleic acid per culture dish or cell, including all values ​​and ranges therebetween. An effective amount of the tumor antigen composition typically ranges from up to, at least, or about 0.01, 0.1, 1, 5, 10 to 10, 15, 20, 50, 100 ng or mg of protein per culture dish or cell. In certain embodiments, 0.001 ng of tumor antigen / cell to 1 μg of tumor antigen / million cells can be used. The tumor antigen composition can be optionally heat inactivated or treated (e.g., exposed to a protease) before contacting with the dendritic cells. Maturation of immature dendritic cells with a nucleic acid composition and a tumor antigen composition allows antigenic priming of mature dendritic cells for a type 1 (Th-1) response.

[0062] Immature DCs are typically contacted with an effective amount of the nucleic acid composition and the tumor antigen composition for at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12-10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 minutes, hours, or days. The immature dendritic cells can be cultured and matured under suitable maturation culture conditions. Suitable tissue culture media include AIM-V (registered trademark), RPMI1640, DMEM, X-VIVO15 (registered trademark), and the like. The tissue culture medium can be supplemented with amino acids, vitamins, as well as cytokines such as GM-CSF and / or IL-4, divalent cations, and the like to promote cell maturation.

[0063] Maturation of dendritic cells can be monitored by methods known in the art. Cell surface markers can be detected by assays well known in the art, such as flow cytometry, immunohistochemistry, etc. Cells can also be monitored for cytokine production (e.g., by ELISA, FACS, or other immunoassays). Dendritic cell precursors, immature dendritic cells, and mature dendritic cells, stimulated or unstimulated by antigen, can be cryopreserved for later use. Methods for cryopreservation are well known in the art. See, for example, U.S. Patent No. 5,788,963, which is incorporated herein by reference in its entirety.

[0064] Nucleic acid or nucleic acid stimulated dendritic cells are dendritic cells that have been incubated or transfected with RNA, such as RNA derived from a tumor or tumor cells. Such RNA can be transfected using conventional nucleic acid transfection methods, such as lipid-mediated transfection, electroporation, and calcium phosphate transfection. For example, RNA can be introduced into DCs by incubating DCs with RNA (or extract) at 37°C for 1 to 24 hours (e.g., 2 hours).

[0065] The nucleic acid-loaded antigen-presenting cells of the present disclosure can be used to stimulate CTL proliferation in vivo or ex vivo. The ability of nucleic acid-loaded dendritic cells to stimulate CTL responses can be measured by analyzing the ability of effector cells to lyse target cells. For example, the commonly used europium release assay can be used. Typically, 5-10x106 target cells are labeled with europium diethylenetriaminepentaacetate for 20 minutes at 4°C. After several washes, serial dilutions of 104 europium-labeled target and effector cells, ranging from effector:target ratios of 50:1 to 6.25:1, are incubated in 200μl RPMI1640 containing 10% heat-inactivated fetal bovine serum in a 96-well plate. The plate is centrifuged at 500xg for 3 minutes and incubated at 37°C, 5% CO2 for 4 hours. A 50 μl aliquot of the supernatant is collected and europium release is measured by time-resolved fluorescence (Volgmann et al., J. Immunol. Methods 119:45-51, 1989).

[0066] A. Genetically Modified Dendritic Cells Certain aspects of the embodiments relate to genetically modified dendritic cells. In some aspects, the genetic modification involves the introduction of an exogenous transgene, such as an inhibitory nucleic acid, into the cells. In further aspects, the transgene can be a suicide gene, such as a gene encoding thymidine kinase, under the control of an inducible promoter. Thus, in some aspects, after stimulating an immune response, the administered dendritic cells can be killed by induction of the promoter that controls the expression of the suicide gene.

[0067] In further embodiments, the genetic modification comprises a genomic deletion or insertion in the cell population. For example, one or more HLA genes can be disrupted to render the dendritic cells an effective HLA match for the subject being treated.

[0068] Further aspects of the embodiment relate to genetically modified dendritic cells, such as to reduce the expression of CTLA-4. In some aspects, the genetic modification comprises introducing an exogenous inhibitory nucleic acid specific for CTLA-4. In certain aspects, the inhibitory nucleic acid is an RNA, such as an RNA expressed from a DNA vector in the dendritic cell. In further aspects, the inhibitory nucleic acid may be an siRNA, dsRNA, miRNA or shRNA introduced into the dendritic cell. A detailed disclosure of such RNA is provided above.

[0069] In further embodiments, the genetic modification comprises a genomic deletion or insertion in the cell population that reduces CTLA-4. In other embodiments, the dendritic cells comprise a hemizygous deletion or homozygous deletion in the CTLA-4 gene. For example, in some embodiments, one or both copies of the CTLA-4 gene in the dendritic cells can be completely or partially deleted, which inhibits the expression of the CTLA-4 polypeptide. In some embodiments, modifying the cells so that they do not express one or more CTLA-4 genes can include introducing an artificial nuclease into the cells that specifically targets the CTLA-4 locus. In various embodiments, the artificial nuclease can be a zinc finger nuclease, a TALEN, or a CRISPR / Cas9. In various embodiments, introducing the artificial nuclease into the cells can include introducing an mRNA that encodes the artificial nuclease into the cells.

[0070] Combination therapy To enhance the effectiveness of dendritic cell therapies involving dendritic cells transformed with nucleic acids produced according to the present embodiments, it may be desirable to combine these compositions with other agents effective in treating the disease of interest.

[0071] As a non-limiting example, the treatment of cancer may be carried out using the antigen stimulatory dendritic cell composition of the present invention in conjunction with other anti-cancer agents. An "anti-cancer" agent may adversely affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit the proliferation of cells. This process may involve contacting the cells with the anti-cancer peptide or nanoparticle complex and the agent(s) or factor(s) simultaneously. This may be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cells with two different compositions or formulations at the same time, one composition including the dendritic cell composition and the other including the second agent(s).

[0072] Treatment with the dendritic cell composition may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the dendritic cell composition are applied separately to the subject, one will generally ensure that no significant period of time lapses between the delivery of each. This allows the agent and the dendritic cell composition to still exert their beneficial combined effect on the cells. In such instances, it is contemplated that both therapies can contact the cells within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the time period for treatment, with several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) passing between each administration.

[0073] When dendritic cell therapy is "A" and a secondary agent such as radiation therapy, chemotherapy or an anti-inflammatory agent is "B", various combinations may be used: A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A In certain embodiments, the administration of the dendritic cell therapy of the present embodiment to patients follows the general protocol for administering chemotherapeutic agents, taking into account the toxicity of the vector (if any). It is expected that the treatment cycle will be repeated as necessary. It is also contemplated that various standard treatments, as well as surgical interventions, may be applied in combination with the described hyperproliferative cell therapy.

[0074] A. Chemotherapy Cancer treatment also includes various combination therapies. In some aspects, the dendritic cell composition of the embodiment is administered (or combined) in combination with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor such as EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitor. Non-limiting examples of protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, benetanib, serotin ... Murafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0075] Still other combination chemotherapy agents include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelanin; acetogenins (especially bullatacin and bullatacinone); camptothecins (e.g., the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (e.g., adozelesin, carzelesin, and the synthetic analog bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; de euocarmycins (synthetic analogs, such as KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma I and calicheamicin omega I1; dynemicins (such as dynemicin A); bisphosphonates, such as clodronate; esperamicin;Neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, multiseromycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, propionate, epithiostanol, mepitiostane, testolactone; antiadrenal such as mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone;Mopidanmol; Nitraline; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine ine);arabinosides ("Ara-C");cyclophosphamide;taxoids such as paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine;platinum;etoposide (VP-16);ifosfamide;mitoxantrone;vincristine;vinorelbine;novantrone;teniposide;edatrexate;daunomycin;aminopterin;xeloda;ibandronate;irinotecan (e.g., CPT-11);topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein tansferase inhibitors, transplatinum, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac (e.g., about 400 to about 800 mg / day of Gleevac may be administered to the patient). In certain embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.;

[0076] B. Radiation therapy Other agents that cause DNA damage and are widely used include gamma radiation, X-rays, and / or what are commonly known as direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also possible, such as microwave and UV radiation. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0077] The terms "contacting" and "exposing" are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell when applied to a cell, e.g., both agents are delivered to a cell in a combined amount effective to kill the cell or prevent the cell from dividing, to achieve cell death or stasis.

[0078] C. Gene Therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the therapeutic composition. Viral vectors for the expression of gene products are well known in the art and include eukaryotic expression systems such as adenoviruses, adeno-associated viruses, retroviruses, herpes viruses, lentiviruses, poxviruses such as vaccinia viruses, and papilloma viruses such as SV40. Alternatively, administration of the expression construct can be achieved using lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, for example, Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0079] D.Surgery Approximately 60% of cancer patients undergo some type of surgery, including preventive, diagnostic, staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatments provided herein, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0080] Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physically removing at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further contemplated that the present embodiments may be used in combination with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue. In some aspects, following tumor resection, the dendritic cell composition of the embodiments is administered to lymphatic tissue draining the former tumor site.

[0081] kit The technology herein includes kits for generating cDNA from mRNA in a sample and for in vitro transcription of cDNA. A "kit" refers to a combination of physical elements. For example, a kit can include one or more components, such as, but not limited to, specific primers, enzymes, reaction buffers, instructions, and other elements useful for carrying out the technology described herein. These physical elements can be arranged in any manner suitable for carrying out the invention.

[0082] The kits may contain, for example, Φ29 polymerase, Bst polymerase, Taq polymerase, Vent polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, 9 oIt may further comprise a DNA polymerase, such as Nm polymerase, T4 polymerase, T7 DNA polymerase, Pfu DNA polymerase, Q5® polymerase (New England Biolabs), or an RNA polymerase, and a reverse transcriptase enzyme, a mutant form of T7 phage DNA polymerase that does not have 3'-5' exonuclease activity, or a mixture thereof.

[0083] The kit may further comprise an RNA polymerase for in vitro transcription, such as, for example, E. coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T7 RNA polymerase, or mutants thereof.

[0084] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components are placed and preferably dispensed appropriately (e.g., into the wells of a microtiter plate). When there are multiple components in the kit, the kit will also typically include a second, third or other additional container into which the additional components can be placed separately. However, various combinations of components may be included in a single vial. The kit of the present invention will also typically include a means for containing the nucleic acid and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are held.

[0085] The kit may also include instructions for using the kit components, as well as the use of any other reagents not included in the kit. The instructions may include implementable variations. Such reagents are considered to be embodiments of the kit of the present invention. However, such kits are not limited to the particular items identified above, but may include any reagent used to manipulate or characterize gene methylation.

[0086] The container means of the kit generally includes at least one vial, test tube, flask, bottle, or other container means into which the components are placed, and preferably suitably dispensed.If there are multiple components in the kit, the kit will also typically include additional containers into which the additional components can be placed separately.However, various combinations of components can be included in the container.The kit of the present invention also typically includes a means for packaging the component containers in close confinement for commercial sale.Such packaging can include injection or blow molded plastic containers into which the desired component containers are held. EXAMPLES

[0087] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes of its practice. However, those of skill in the art should, in light of this disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0088] Example 1 - Materials and Methods Fresh tissue processing - After surgical removal of the tumor, the tumor tissue was weighed and sliced ​​into small pieces (approximately 20 mg / piece or less). All pieces were placed in a 1.5 mL cryovial containing 1 mL of RNA stabilizing solution (RNALATER™, Invitrogen). The cryovial containing the RNALATER™ and dissected tumor was left overnight at 4°C to prevent RNase activity. After overnight incubation at 4°C, the cryovial containing the RNALATER™ solution containing the tissue was centrifuged at 10,000 RPM for 1 minute at 4°C. After centrifugation, the supernatant was removed by pipetting and the tissue pieces were weighed. 10% of the tissue was used for the subsequent preparation of total RNA and the remaining 90% was used for the subsequent preparation of antigen lysates.

[0089] Preparation of antigen lysate - Dissected tumor tissue was added to a 15mL conical tube and diluted to 100mg / mL in sterile PBS. A homogenous suspension of tissue in PBS was then made using a handheld tissue homogenizer. The suspension was then frozen by immersing the tube with the homogenous suspension in a liquid nitrogen or dry ice / ethanol bath. After freezing, the cell suspension was incubated in a 55°C water bath until completely thawed. This freeze-thaw cycle was repeated two more times. After the final thaw, the cell suspension was gamma irradiated to >2500Gy via a cesium irradiator to ensure sterility and complete cell death. The lysate was then stored at -20°C until further use.

[0090] Antigen mRNA Preparation - Total RNA was isolated from tissues stabilized with RNALATER™ Reagent using the RNEASY® Mini Kit (Qiagen Cat. No. 74104) utilizing spin columns or the TRIPURE® (Roche) method utilizing a guanidinium thiocyanate and phenol mixture, each according to the manufacturer's instructions.

[0091] Preparation of RNeasy total RNA - 600 μL of Buffer RLT was added to 30 mg of tissue and the resulting mixture was homogenized using a handheld tissue homogenizer. 600 μL of 70% ethanol was added to the lysate and mixed by pipetting. 700 μL of the sample was transferred to an RNeasy spin column and placed in a 2 mL collection tube. The sample was centrifuged at 8,000 x g for 1 min. After centrifugation, the flow-through was discarded. The remaining lysate was added to the spin column and centrifuged again. After centrifugation, the flow-through was discarded. 350 μL of Buffer RW1 was added to the column and centrifuged. DNase I solution was prepared by adding 10 μL of DNase I stock solution to 70 μL of Buffer RDD followed by gentle mixing. Then, 80 μL of DNase I solution was added to the column and incubated at room temperature for 15 min. After DNase I incubation, 350 μL of Buffer RW1 was added to the column and the column was centrifuged at 8000xg for 1 min. After centrifugation, the flow-through was discarded. The membrane was then washed by adding another 700 μL of Buffer RW1 and centrifuged at 8000xg for another 1 min, followed by washing twice with Buffer RPE with 500 μl and centrifuging twice at 8000xg. The membrane was then dried by centrifugation for 2 min. The RNA was eluted into a new column by adding 50 μl of pre-warmed nuclease-free water directly to the membrane in the column, incubating for 1 min, and centrifuging at 8000xg for 1 min. A second elution was performed using the eluent to concentrate the RNA. Total RNA was then quantified. Total RNA was stored at -80 °C for long-term storage.

[0092] Guanidinium thiocyanate / phenol / chloroform RNA isolation - For every 50 mg of tumor tissue, 1 mL of guanidinium thiocyanate and phenol reagent (TRIpure™ reagent (Roche), catalog number 11 667 157 001) was added and the resulting mixture was homogenized using a handheld tissue homogenizer. The homogenized mixture was then centrifuged at 13,000 RPM for 5 minutes at 4°C to remove the homogenate. The homogenate supernatant was transferred to a new RNase-free tube. To remove the supernatant, 400 uL of chloroform was added and the mixture was mixed thoroughly by inversion. The supernatant / chloroform mixture was then incubated at room temperature for 5-10 minutes. The mixture was then centrifuged at 4500 RPM for 45 minutes or 12,000 x g for 15 minutes at 4°C to remove the supernatant. After centrifugation, the resulting mixture exists in three phases (organic phase, interface, and aqueous phase). The aqueous phase was collected by pipetting and transferred directly to a pre-chilled tube. For every 1 mL of guanidinium thiocyanate and phenol reagent starting material, 0.5 mL of isopropanol was added. The isopropanol / supernatant mixture was mixed gently and incubated at room temperature for 10 minutes. After incubation, the RNA was pelleted by centrifugation at 4500 RPM for 45 minutes or 12000xG for 15 minutes at 4°C. The pelleted RNA was washed with 1 mL of 70% ethanol and centrifuged again. The supernatant was removed by pipetting and the pellet was allowed to air dry on ice for 5-10 minutes. The pellet was then dissolved in 100 uL of RNase-free water and incubated at 55°C for 5 minutes. The concentration of total RNA was then measured. Total RNA was stored at -80°C for long-term storage. Prior to first strand synthesis, total RNA isolated using the guanidinium thiocyanate method was DNase treated by adding 1 uL of amplification grade DNase (Invitrogen Catalog No. 18068-015) to 10X buffer diluted to 1X containing 4ug of total RNA. The DNase reaction was allowed to proceed for 15 minutes and stopped by adding 1 uL of EDTA.

[0093] cDNA Synthesis - For each sample and control, the following RNA / primer annealing mix was prepared in a sterile 0.2 mL PCR tube: 4 uL of a 10 uM solution of SEQ ID NO:1 (VKWD oligos), 4 uL of a 10 uM solution of SEQ ID NO:2 (CDS64T+ oligos), 4 ug of sample RNA, and water for a total reaction volume of 19 uL. The solution was mixed and spun briefly in a microfuge to condense the solution to the bottom of the tube. This solution was then incubated at 72°C for 2 minutes in a preheated thermocycler and cooled to 4°C. Samples were then stored on ice while the first strand synthesis master mix was prepared.

[0094] A first strand synthesis master mix was prepared for each reaction plus one no-template control from the reagents in the Superscript® First Strand Synthesis Kit (Invitrogen Catalog No. 11904-018). The master mix included 4 uL of 10X First-Strand Buffer, 4 uL of DTT (100 mM), 1 uL of RNase inhibitor (RNase Out™), 4 uL of MgCl2 (25 mM), 4 uL of dNTP mix (10 mM each of dATP, dGTP, dCTP, and dTTP), and 4 uL of Superscript II Reverse Transcriptase® per sample for a total volume of 21 μL per sample.

[0095] 21 uL of First Strand Synthesis Master Mix was added to each tube containing 19 ul of RNA / Primer Annealing Mix. Each reaction, such as NTC, was incubated at 42°C for 1 hour to synthesize the first strand of cDNA. The first strand synthesis reactions were then collected by brief centrifugation in a microfuge and chilled on ice before synthesizing second strand cDNA.

[0096] A second strand synthesis master mix was prepared by adding 100 uL FastStart™ High Fidelity Reaction Buffer with MgCl2, 40 uL of 10 μmol / L CDS64T+ Oligo (SEQ ID NO:2), 20 uL of 20 μmol / L Powerswitch T7 Primer (SEQ ID NO:3), 20 uL dNTP mix (10 mM each of dATP, dGTP, dCTP, dTTP), 20 uL FastStart High Fidelity Enzyme, and 780 uL of water (total of 980 uL / sample). 20 uL of the first strand synthesis reaction was added to the second strand synthesis master mix. The resulting mixture was then split into 10 equal portions of 100 uL each in 0.2 mL PCR tubes. Each of these reactions was subjected to thermal cycling using the following program: 1x 95℃, 5 min 11x 95℃, 30 seconds 65℃, 30 seconds 67°C, 6 min 9x 95℃, 35 seconds 65℃, 35 seconds 67℃, 6 minutes 5 seconds For each additional cycle, the extension at 67° C. is increased by an additional 5 sec. e.g., cycle 1=6:05, cycle 2=6:10, cycle 3=6:15, etc. 1x 67℃, 7 min Hold at 4°C.

[0097] The double-stranded cDNA was then purified using salt-ethanol precipitation. Sodium acetate (3M) was added to the sample in a 1:10 volume ratio (vol:vol), followed by chilled 100% ethanol in a 2:1 volume ratio (vol:vol). The sample was gently mixed and incubated at -20°C for 1 hour. The mixture was then centrifuged at 13,000 RPM for 20 minutes to pellet the DNA. The supernatant was carefully pipetted off the pelleted DNA. The resulting pellet was then washed and resuspended by adding 100uL of chilled 70% ethanol. The solution was then centrifuged at 13,000 RPM for 10 minutes, and the supernatant was pipetted off. The pellet was then air-dried for 10 minutes before being dissolved in 20uL of nuclease-free water. The concentration of the cDNA was measured with a Nanodrop.

[0098] In vitro transcription - In vitro transcription was performed using the AMPLISCRIBE™ T7-FLASH® in vitro amplification kit (LUCIGEN™ catalog number ASF3257). A 20 uL reaction volume was prepared by adding in the following order: X uL RNase-free water (X is the amount of water needed to achieve a 20 uL reaction volume); 1 ug cDNA template; 2 uL AMPLISCRIBE™ T7-FLASH® 10X reaction buffer; 1.8 uL each of 100 mM ATP, CTP, GTP, and UTP; 2 uL DTT; 0.5 uL RIBOGUARD™ RNase inhibitor; and 2 uL AMPLISCRIBE™ T7-FLASH® enzyme solution. The reaction was then incubated in a thermocycler at 42°C for 1 hour. After incubation, 1 uL RNase-free DNase I was added and the reaction was incubated at 37°C for 15 minutes. The DNase-treated in vitro transcription reaction was brought to 50 uL by adding 29 uL of RNase-free water. The in vitro transcription reaction was then purified by salt and ethanol precipitation and purification. 50 μL of 5 M ammonium acetate and 100 uL of ice-cold 100% ethanol were added to the in vitro transcription reaction and mixed. The resulting mixture was then incubated at -20°C for 1 hour. After incubation, the mixture was centrifuged at 10,000xg for 15 minutes at 4°C. The supernatant was removed from the pellet by pipetting. The pellet was then washed with 100 uL of cold 70% ethanol and centrifuged again at 10,000xg for 15 minutes at 4°C. The supernatant was removed by pipetting and the pellet was air-dried before being resuspended in 50 uL of RNase-free water.

[0099] Capping of uncapped in vitro transcribed RNA - Capping of in vitro transcribed RNA was performed using the SCRIPTCAP™ m7G Capping System and SCRIPTCAP™ 2'-O-Methyltransferase Kit (CELLSCRIPT™ Catalog Nos. C-SCCE0625 and C-SCMT0625) according to the manufacturer's instructions. Briefly, 50-60ug of in vitro transcribed uncapped RNA was added to RNase-free water to a total volume of 67uL. The diluted RNA was then incubated at 65°C for 10 minutes to denature and then stored on ice. Capping master mix was prepared by adding 10uL 10X SCRIPTCAP™ Capping Buffer, 10uL 10mM GTP, 2.5uL 20mM S-Adenosylmethionine, 2.5uL SCRIPTGUARD™ RNase Inhibitor, and 4uL SCRIPTCAP™ 2'-O Methyltransferase per sample. Capping reactions were assembled by adding 4uL SCRIPTCAP™ Capping Enzyme to 29uL Capping Master Mix and adding the mix to the heat denatured RNA. Capping reactions were allowed to proceed for 30 minutes at 37°C. Capped RNA was purified by salt and ethanol precipitation and purification as above and resuspended in 50uL RNase-free water. To assess the quality of the capped mRNA, 1ug ​​of capped mRNA was run on a 50mL 1% agarose non-denaturing gel. Capped mRNA was stored at -80°C.

[0100] Preparation of antigenic lysates - To generate MHC class II determinants, 1 mL of PBS per 100 mg of tissue was added to the isolated tissue fractions and disrupted using a POLYTRON™ PT1200E tissue homogenizer (Kinematica, Inc., Bohemia, New York). The homogenized tissue fractions were then subjected to three repeated freeze-thaw cycles, and the homogenized mixture was cycled between liquid nitrogen and a 55°C water bath and stored at -20°C.

[0101] Human (Decker et al., 2006; Decker et al., 2009) and wild-type mouse (Konduri et al., 2013) dendritic cells were prepared, loaded, and matured as described. In vitro cocultures were performed as previously described (Decker et al., 2006; Decker et al., 2009; Konduri et al., 2013).

[0102] IFNγ production by T cells co-cultured with dendritic cells loaded with capped mRNA. Total PBMCs in the co-culture were treated with 10 μg / ml brefeldin A (eBioscience) for 5 h. Cells were stained for surface markers with anti-CD3, anti-CD8, and anti-CD25 antibodies and fixed / permeabilized for intracellular staining of IFN-γ using Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer's instructions.

[0103] Loading with homologous antigen increases AIMp1 production and retains CTLA-4. Increased production of AIMp1 was demonstrated by Western blot. Increased retention of CTLA-4 was demonstrated by CTLA-4 Western blot of cell culture supernatants (to determine release) and intracellular flow cytometry to determine retained CTLA-4 content. Histogram analysis of intracellular CTLA-4 is based on CD11c+CD80+CD83+CD86+ cell population.

[0104] Western blot analysis. Preparation of whole cell lysates: Cells were lysed on ice with 1% NP-40 lysis buffer containing protease inhibitor cocktail, phosphatase inhibitor cocktail 2 and phosphatase inhibitor cocktail 3 (all purchased from Sigma-Aldrich) and vortexed every 10–15 min. Cell lysates were centrifuged at 14,000xg for 15 min and the cleared lysates were denatured in Laemmli buffer (Bio-Rad) containing 5% β-mercaptoethanol (Bio-Rad) for 10 min. Denatured whole cell lysate samples were stored at -20 °C for further analysis. Electrophoresis and blotting: Protein samples were separated by SDS gel electrophoresis (Invitrogen) and then transferred to 0.45 μm nitrocellulose membranes (Bio-Rad) for antibody probing. All blocking and antibody staining steps were performed in 5% BSA (RPI, Grainger) / 1xTBST buffer (0.05% Tween-20). Western blot chemiluminescence signals were detected with SUPERSIGNAL WestFemto Maximum Sensitivity Substrate (ThermoFisher Scientific) using a CHEMIDOC® XRS digital imaging system supported by Image Lab software version 2.0.1 (Bio-Rad). Densitometry was performed using Image Lab software.

[0105] Example 2 - Results RNA isolation from fresh tissue yields higher quality RNA - To determine whether fresh or frozen tissue produced higher quality RNA in downstream processing, total RNA was isolated from 30 mg of three different frozen tumor tissues or a single fresh tumor. As seen in Figure 1, RNA isolated from the three frozen tissues exhibits significant degradation, visible as a large smear in the lanes. Lane 4 has two clearly defined bands, indicating high RNA integrity.

[0106] Comparison of commercial RNA isolation methods - Total RNA was isolated from 30 mg of tumor tissue using either the RNEASY® Mini Kit (Qiagen) or the TRIPURE® RNA Isolation Reagent (Roche), a guanidinium thiocyanate / phenol / chloroform based method. Both of these methods yielded high quality RNA as seen in Figure 2. However, the TRIPURE® method yielded 4.4-fold more RNA than the RNEASY® Kit with the same input amount.

[0107] In Vitro Transcribed RNA from mRNA Isolated from Tumor Cells - Figure 3 shows a diagram of the primers etc. for the method (see Example 1) used for reverse transcription of mRNA isolated from tumor cells and subsequent in vitro transcription of cDNA by the method of the invention. Two different reverse transcription and in vitro transcription methods were compared (Figure 4). Lanes 1 and 2 show either uncapped or capped RNA prepared by the Slagter-Jager method (Slagter-Jager et al., 2013). Lanes 3 and 4 show either uncapped or capped RNA prepared by the method described herein. Whereas previous methods showed enrichment of very small transcripts, the method described herein resulted in a smear of RNA at larger molecular weights.

[0108] Enrichment of small transcripts by co-transcriptional capping. To ensure accurate representation of all transcripts, two methods were tested for capping of in vitro transcribed mRNA. Figure 5 shows an agarose gel of capped RNA generated by each method. Lane 1 shows RNA that is capped after transcription, and lane 2 shows RNA that is capped after co-transcription. Clearly, in lane 2, the transcriptional smear is much more evident, indicating that RNA generated by the current method is capped by co-transcription. Tables 1 and 2 show the results of the Slagter-Jager method (Slagter-Jager The concentrations, volumes, and yields of the products produced by the method described above are shown. The preparation of the Konduri / Decker hybrid follows this method.

[0109] [Table 1]

[0110] [Table 2]

[0111] IFNγ production is increased by co-culture with loaded DCs - dendritic cells were either homologously loaded with antigen lysate and mRNA prepared as described herein, or heterologously loaded with prostate mRNA and pancreatic tumor lysate (mismatched). Figure 6 shows the relative IFNγ levels produced by T cells co-cultured with the indicated dendritic cells. Dendritic cells loaded with capped RNA and antigen lysate clearly stimulated IFNγ production, superior to dendritic cells loaded with uncapped RNA, or immature or unloaded dendritic cells.

[0112] Coculture with homologously loaded dendritic cells resulted in antigen-specific CD8 + CD25 + T cells are expanded - FIG. 7 shows CD8 T cells sorted by IFNγ levels after co-culture with dendritic cells either homologously loaded with uncapped or capped mRNA and lysates produced by the methods described herein, or loaded with prostate mRNA and pancreatic tumor lysate (mismatched). + CD25 + The results of flow cytometry analysis of cells are shown. Homologously loaded lysates and mRNA produced by the methods described herein were able to express antigen-specific CD8 + CD25 + The levels of T cells are significantly increased.

[0113] Homologous antigen loading increases AIMp1 production and CTLA4 retention - Dendritic cells were assessed for AIMp1 production and CTLA4 retention after loading with homologous antigen or heterologous antigen (mismatched). Figure 8 shows a Western blot of AIMp1 and CTLA-4 in lysate with β-actin provided as a control. Loading of homologous antigen with capped mRNA produced by the methods provided herein increases the amount of AIMpl and the amount of intracellular CTLA-4 as seen in lane 5. To confirm this, flow cytometry was performed on dendritic cells either unloaded, loaded with only mRNA, lysate only, both, or with mismatched antigen. Figure 9 shows that CTLA4 retention is significantly increased in CD11c+CD80+CD83+CD86+ cells loaded with both mRNA and homologous antigen lysate. Enhanced retention of CTLA4 was further confirmed in cells homologously loaded with antigen lysate and mRNA produced by the methods provided herein by probing dendritic cell lysates and supernatants for CTLA4 by Western blot (FIG. 10). Loading with antigen lysate and mRNA prepared by either method reduced the amount of CTLA4 present in the supernatant, indicating enhanced retention.

[0114] Homologous antigen loading increases IL-12 transcription in dendritic cells - dendritic cells were assessed for IL-12a and IL-12b production by RT-qPCR. As seen in Figure 11, antigen loading with both lysate and mRNA produced by the methods provided herein (TRIpure-IVT) increases both IL-12 transcripts more than loading with lysate, mRNA, or heterologous loading alone, and also increases the production of IL-12 transcripts more than loading with mRNA prepared by previous methods (column-IVT).

[0115] Example 3 - Further studies with amplified mRNA Additional studies have shown that amplified mRNA, when loaded into DCs together with homologous lysates, generates TH1 immune responses in vitro with the same efficiency as native polyA mRNA. For example, results shown in FIG. 13A show monocyte-derived human DCs loaded with a) native polyA tumor mRNA and homologous / heterologous lysates, or 2) capped IVT-amplified mRNA and homologous / heterologous lysates. DCs were then co-cultured with T cells and the percentage of activated CD8+CD25+Ifng+ cells was analyzed by flow cytometry. T cells co-cultured with unloaded DCs or with uncapped mRNA-loaded DCs served as controls. FIG. 13B shows DCs loaded with various combinations of polyA mRNA or IVT-amplified mRNA and homologous or heterologous cell lysates were matured for 48 hours and analyzed for intracellular CTLA4 levels by flow cytometry. Singly loaded DCs and unloaded DCs served as controls. Upregulation of intracellular CTLA-4 indicates increased retention and therefore decreased secretion.UL-unloaded DCs, uncapped mRNA-uncapped IVT mRNA loaded DCs, mRNA-polyA mRNA loaded DCs, lysate-lysate loaded DCs, 2xw / uncapped-uncapped IVT mRNA and homologous lysate loaded DCs, 2xcapped-capped IVT mRNA and homologous lysate loaded DCs, uncapped MM-uncapped IVT mRNA and heterologous lysate loaded DCs, capped MM-capped IVT mRNA and heterologous lysate loaded DCs.

[0116] The method of the embodiment was able to amplify sufficient amounts of mRNA from very small amounts of starting tissue for use in stimulating an immune response. The results shown in Table 3 below show the amount of RNA that could be produced from various starting samples.

[0117] [Table 3]

[0118] All of the methods disclosed and claimed herein can be made and accomplished without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied in the methods and steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, or scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references, to the extent that they provide exemplary procedural or other detail supplementary to those set forth herein, are specifically incorporated herein by reference. Slagter-Jager et al. “Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response”Mol Ther Nucleic Acids,2(5):e91,2013

Claims

[Claim 1] The invention described in the specification.

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