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  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/80—Vaccine for a specifically defined cancer
  • A61K2039/804—Blood cells [leukemia, lymphoma]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

• the invention relates generally to vaccines and particularly to cancer vaccines prepared by either transfection of cancer cells or direct intratumoral administration with a synthetic bacterial messenger ribonucleic acid (mRNA).
• mRNA synthetic bacterial messenger ribonucleic acid
• the invention more particularly describes synthetic RNAs that efficiently express a selected polypeptide in mammalian cells and the use of the RNAs to transform the cells in vivo or in vitro.
• the present invention provides for development and use of effective mRNA vaccines for cancer treatment.
• deoxyribonucleic acid (DNA) vaccines have several deficiencies, including low transfection efficiency and time consuming delivery methods
• the mRNA vaccines of the present invention are administered directly into tumor cells and immediately translated into an immunogenic protein which evokes a multi-tumor-antigen response.
• the short in vivo half-life of mRNA makes it less likely to integrate into the host genome compared to plasmid DNA and is therefore considered safer.
• mRNA- vaccines do not need to cross the nuclear envelope and thus can often generate more rapid and higher levels of protein expression.
• mRNA vaccines lack Major Histocompatibility Complex (MHC) haplotype restriction and can be designed to be self-adjuvating with the addition of MHC I trafficking signal or by combination with protamine.
• MHC Major Histocompatibility Complex
• the efficacy of mRNA vaccines may benefit from complexing agents which protect RNA from degradation and enhance cellular uptake, cells in vivo can be transfected with mRNA in the absence of other reagents or physical transduction methods.
• Messenger RNA for use as a vaccine can be generated from plasmid DNA using in vitro transcription.
• Treatment for cancers is based on the specific type that is diagnosed. Some common cancers include bladder, breast, colon, lymphoma, melanoma and prostate. Treatment regimens are prepared by physicians based on the evaluation of multiple factors including, but not limited to, disease stage, etiology and patient age and general health. For many cancers the treatment regimen can include one or a combination of surgery, chemotherapy, radiation, bone marrow/stem cell transplants, cancer drugs or immunotherapy. The most common treatments include surgery, chemotherapy, radiation and oral drugs. Although these treatments can be effective there are often many side effects. Chemotherapy in particular targets all newly dividing cells in the body not just the cancerous cells.
• the advantage of some immunotherapies is the ability to target the diseased cells while leaving the non-diseased cells intact. Cancerous cells arise from a breakdown in normal growth regulatory mechanisms; therefore, the body still sees many of these cells as self. Cancer immunotherapy overcomes the body’s tolerance of these diseased self-cells and allows the body to distinguish them as foreign. Cancer can also escape immune detection through direct suppression of the body’s immune system by decreasing expression of immune activating markers on cells such as MHC molecules.
• the MHC is one of the components that help the body differentiate which cells are self and which are foreign or diseased.
• Treatments for solid cancers typically include chemotherapy and/or surgery. Recently there has been interest in developing vaccines in an effort to stimulate an autologous immune defense.
• U.S. Patent No.7,795,020 describes in detail a lymphoma vaccine for treating advanced stages of lymphoma with transformed autologous or non-autologous cells isolated from a subject diagnosed with lymphoma. The isolated cells are transfected with a plasmid vector carrying a Streptococcus pyogenes emm55 gene. The bacterial protein is expressed on the cell surface and when the transfected cells are introduced to a subject with the cancer, generates an immunological response to lymphoma cells.
• Biovaxld is an autologous tumor derived immunoglobulin idiotype vaccine undergoing Phase III clinical studies in the treatment of indolent follicular Non- Hodgkin Lymphoma.
• RNA can express proteins in the mammalian body. Whether or not similar immune activity can be produced with both DNA and mRNA expressed proteins is uncertain. Conventional wisdom is that DNA is superior for the creation of vaccines and gene therapy due to its stability and ease of use.
• An example of a plasmid DNA vaccine is MeriaTs Oncept, which was developed for treatment of oral canine melanoma.
• FIG. 3 is a table of published clinical trials using mRNA vaccines.
• Delivery vehicles such as liposomes and cationic polymers appear to have promise in enhancing transfection. Once the liposome or polymer complex enters the cytoplasm, the mRNA must be able to separate from the delivery vehicle to enable antigen translation; unfortunately, these vehicles may not properly complex with mRNA and therefore not allow for proper translation of the encoded protein. Antigen production may occur but in amounts insufficient to produce a desired effect.
• Many immunotherapies are disease-specific, complicated in concept and even more complicated and expensive to produce. It remains to be seen whether such therapies will be commercially viable. The administration of mRNA directly into a patient’s tumor where it is immediately translated into an immunogenic protein which evokes a multi-tumor-antigen response has far-reaching implications.
• mRNA For instance, a single synthetic mRNA can be used to treat multiple types of cancer in multiple species. mRNA is simple to deliver, cost-effective, easily transported and stored, as well as easy to administer. Along with an excellent safety profile, these attributes of mRNA make it possible to treat cancer patients worldwide, even in developing countries.
• nucleic acid vaccines can induce both humoral and cellular immune responses; have low effective dosages; are simple to manipulate; avail rapid testing; are cost-effective and reproducible in large scale production and isolation; can be produced at high frequency and are easily isolated; are more temperature-stable than conventional vaccines; have a long shelf-life; are easy to store and transport; and are unlikely to require a cold chain.
• DNA has been used in vaccines with success.
• DNA is a double stranded molecule that serves as the blueprint, i.e., genetic instructions, for organisms.
• DNA is amenable to use as a vaccine as it is fairly stable and unreactive and can be stored long term.
• DNA is self-replicating and can be easily damaged by ultra-violet radiation.
• RNA is single stranded and functions to carry out the DNA’s instructions, i.e., RNA transfers the genetic code to create proteins.
• RNA is more reactive than DNA and less stable but is resistant to ultra-violet radiation. As it turns out, these latter qualities make RNA better suited to use as vaccines.
• mRNA has zero chance of integrating into the host chromosomes. The delivery of mRNA results in faster expression of the antigen of interest and requires fewer copies for expression. mRNA expression is transient, which seems like a disadvantage but actually adds to its safety.
• mRNA is more effective than DNA for protein production in post mitotic and non-dividing cells because DNA requires translocation through the nuclear member and plasmid membrane, while mRNA requires translocation only through the plasmid membrane.
• mRNA is not only a template for translation, but also acts as a ligand for toll-like receptors and is nuclease sensitive; therefore it presents less concern for horizontal transmission.
• the invention is based on use of a ribonucleic acid message (mRNA) (SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16) that encodes an immunogenic bacterial protein.
• mRNA ribonucleic acid message
• the message can be delivered into the cell cytoplasm using any of a number of known techniques. Once the mRNA reaches the cytoplasm, it is translated into the encoded protein using the cellular machinery already in place.
• the bacterial protein such as an M-like protein having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 14, will then be expressed in the cell rendering immunogenicity to a cancer cell.
• M-like proteins can be derived from the bacterial sources, Group A and G Streptococci (GAS and GGS), and therefore are seen by the mammalian body as foreign.
• Immune monitoring cells such as antigen presenting cells (APCs) are attracted by the foreign protein.
• the APC’s will phagocytize the entire cancer cell and then present all the foreign/mutant proteins including M-like protein to other immune cells.
• the gene for M-like proteins is referred to as emmL.
• emmL The gene for M-like proteins.
• the emmL message is delivered into a cancer cell containing abnormal proteins produced by the mutation in the cell’s DNA, the M-like protein will be expressed in the cell, attracting immune cells to engulf it, and lead to the presentation of the previously-masked mutated proteins to the immune system.
• Abnormal proteins may have been present for an extended period of time, but because they were derived from “self’ proteins, the body would not necessarily view them as foreign or a threat.
• the bacterial protein antigen acts as a primer or trigger for the immune system to address cells that it otherwise may not have been able to identify previously as damaged and harmful.
• the mRNA is produced as described in the examples. Once obtained, the mRNA containing the immunogenic message can be delivered into autologous or allogeneic cells that require the priming affect described in the summary of invention. The mRNA can also be directly delivered intratumorally or in the case of some cancers such as lymphoma, intranodally as well.
• RNA messenger RNA
• mRNA expression lasts only a few hours to a few days at maximum inside a cell. mRNA that is not delivered into the cells is quickly degraded by RNases that are present in the environment and therefore does not pose the risk of being horizontally transmitted.
• FIG. 1 illustrates the plasmid used for the backbone of the recombinant plasmid designed for mRNA production of M-like protein.
• FIG. 2 illustrates the plasmid DNA used as the source for the emmL gene to be ligated into the linearized vector of FIG. 1.
• FIG. 3 is a summary of mRNA trials performed in solid cancers.
• FIG. 4 is a diagram demonstrating the cellular production pathway differences between mRNA and DNA.
• FIG. 5 shows the creation of a recombinant DNA vector to produce an mRNA encoding a bacterial antigen.
• FIG. 6 shows a Western blot of isolated Emm55 against an anti-M-like antibody.
• FIG. 7 shows comparative results of protein expression from DNA and RNA transfection.
• FIG. 8 is a photograph of an agarose gel of synthesized emm55 mRNA using a Flashgel system.
• FIG. 9 is a graph showing antibodies reactive to EmmL protein in mice vaccinated with emmL mRNA or water (controls) (C2) after weeks 1, 2, 3 and 4.
• FIG. 10 is a Western blot showing presence of antibodies in mice blood that react to EmmL protein pre and post vaccination with emmL mRNA.
• FIG. 11 is the map of a novel double stranded DNA molecule used in the production of synthetic mRNA that can be translated at high efficiency in mammalian cells.
• This DNA molecule contains the sequences for T7 RNA polymerase (SEQ ID NO: 8), a portion of the Xenopus laevis beta globin gene 5’ untranslated region (SEQ ID NO: 9), a polylinker with restriction endonuclease recognition sites for Sad, Notl, Bglll, EcoRV and Spel used to insert the coding region of emmL (SEQ ID NO: 7), a portion of the Xenopus laevis beta globin gene 3’ untranslated region (SEQ ID NO: 10), then a polylinker with restriction endonuclease recognition sites for BamHI, EcoRI and Xbal used to linearize the plasmid prior to in vitro transcription (SEQ ID NO: 11).
• the DNA sequence corresponding to Figure 11 is SEQ ID NO: 12.
• FIGs. 12A-12B show relative adaptiveness plot of emm55.
• Y axis is the adaptiveness index for each codon of emm55, where 1.0 is perfectly adapted for human cell expression.
• FIG. 12A is the original emm55 sequence.
• FIG. 12B shows emm55 after optimization with the JCat algorithm.
• FIGs. 13A-13C are simulated western blots from WesTM capillary electrophoresis analysis of Emm55 expressed following transient transfection of HEK293T and B16-F10 cells with wild type uridine (WT) and N1 -methyl pseudouridine containing mRNA. Lysates were at 0.5 pg/pL. ERK1 used as a normalizing control. -, cells transfected with EGFP mRNA. *, 6 hr post Emm55FreqDist transfection HEK293T cell lysate incubated with rabbit anti-ERKl only.
• FIG. 13A is emm55Jcat.
• FIG. 13B is emm55MostFreq.
• FIG. 13C is emm55FreqDist. *, 6 hr post Emm55FreqDist transfection HEK293T cell lysate incubated with rabbit anti-ERKl only.
• FIGs. 14A-14B show the normalized expression of Emm55 from images shown in Figure 13. Codon Adaptiveness Index is used to determine codon optimization in the JCat algorithm, where 1.0 is perfectly optimized.
• FIG. 14A shows expression in HEK293T cells.
• FIG. 14B shows expression in B16-F10 cells.
• FIGs. 15A-15B show the time course of Emm55 expression in HEK293T cells.
• FIG. 15A is the initial assay performed at the WesTM maximum signal intensity.
• the present invention provides a cancer vaccine that can be prepared efficiently and with less expense than previously used vaccines, which are prepared by introducing plasmid DNA directly into the nucleus of the cell.
• the use of an emmL encoding mRNA (SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16) inserted into the cell cytoplasm is more effective in transfecting tumor cells.
• the present invention further provides several new emmL encoding RNAs designed to optimize increased expression in transformed cells.
• the more robust expression of Emm55 protein was achieved using several codon optimization algorithms designed to adapt the emm55 nucleotide sequence to mammalian expression.
• mRNA is capable of antigenic protein message delivery into cancer cells. Not only is mRNA a safer alternative because it cannot integrate into the host DNA, but also expression is limited to only a few hours to a few days at maximum. mRNA delivery also places the antigenic message further down the cellular protein production process, therefore providing a quicker expression in the cells. The mRNA only has to be delivered into the cell cytoplasm, whereas DNA must ultimately end up in the nucleus to be effective. The use of mRNA is an advantage for production of protein antigen because mRNA can induce protein production in both post mitotic and non-dividing cells.
• mRNA delivery of antigenic proteins is a safer alternative to DNA delivery, stability and immunogenicity of mRNA must be addressed.
• Many of the elements that facilitate increased stability and immunogenicity were engineered into the recombinant vector template. If appropriate elements are not contained in the vector, they can be added; for example, if the template vector does not contain a poly(A) tail coding sequence, the tail is added during transcription.
• the mRNA In order to have increased stability in the cytoplasm, the mRNA must contain both a 5’-methylguanosine cap and a 3’-poly(A) tail (SEQ ID NO: 3). These elements are responsible for attracting and attaching the components of the cell machinery responsible for translating the mRNA into proteins. Absence of these components can decrease the time in which the mRNA is available for protein translation prior to degradation. Accordingly, these elements have been incorporated into the mRNA as described in the examples.
• Efficient immunogenicity can be increased by utilization of techniques such as enhanced delivery with viral vectors, nanoparticles, cationic polymers, lipids and electroporation.
• Viral vectors have been used extensively in delivery of plasmid DNA (pDNA) but have several risks, as well as increased cost.
• Electroporation with mRNA is less toxic to cells due to less stringent electrical settings and is a preferable method for delivery of mRNA.
• DNA requires a higher electrical charge to pass the DNA through the outer cellular membrane and the nuclear membrane, while mRNA needs to pass only though the outer cellular membrane.
• mRNA for vaccines has both economic and production benefits compared to pDNA.
• mRNA is synthesized in vitro from a linearized pDNA template and only a small amount of DNA is required.
• production of large amounts of pDNA is labor intensive and requires equipment such as large fermentation tanks to grow sufficient bacteria to produce the massive amount of pDNA required for vaccine production.
• pDNA isolated from large cultures is pure, due to the circular nature of plasmids the end product occurs in three structural forms; relaxed, linear and supercoiled. Although each form has the ability to produce an antigenic protein once inside a cell, each DNA form varies in its ability to enter the cell via the plasma membrane. Production of mRNA creates only one structural form. Moreover, due to the synthetic method of production the batch to batch reproducibility is high.
• mRNA is synthesized from DNA and is highly reproducible. This is important for use as a vaccine because no large scale growth is required, i.e. it takes less time and materials and there is less risk of contamination. These factors contribute to reduced costs. Further, synthesis of mRNA leads to higher yield since it only takes one linearized plasmid DNA to yield one hundred mRNA molecules. mRNA is produced in vitro , so there is no E. coli contamination post isolation (genomic DNA or endotoxin). This leads to fewer purification steps and quality control tests. The synthetic nature of in vitro transcription also ensures better batch to batch reproducibility and purer product since vector sequences, including selection markers, are not part of final product.
• mRNA has a single molecular conformation, whereas plasmid DNA has three. mRNA is also easier to transfect than plasmid DNA and results in less cell death during electroporation since lower voltage is required. Like DNA, mRNA can also be lyophilized. From a regulatory standpoint, mRNA is safer because it is non-replicating and is transient. mRNA also poses minimal to no environmental issues since it is easily degraded and confers no antibiotic resistance.
• the below comparison illustrates the advantages of using mRNA instead of DNA for antigenic emmL message delivery into cancer cells.
• the comparison is broken up into three parts; upstream production, downstream production and cellular delivery.
• the bulk of the benefits, including decreased production cost, reduced manufacturing time, superior message delivery and increased safety, are seen in the upstream production and cellular delivery.
• Each section shows a large difference between the DNA and mRNA processes as well as similar steps for each process.
• the benefit of having to grow only a small bacterial culture is significant.
• the small amount of DNA from this culture requires a smaller isolation to be performed. This downsizing saves time and resources and decreases contamination risk.
• the production of the final mRNA product requires an additional step of transcribing the mRNA from the DNA template. This is a synthetic step performed in vitro. Due to the synthetic nature of transcription, there is good batch to batch reproducibility and the procedure takes only a few hours. Culturing DNA-containing bacteria can require up to several days.
• a significant disadvantage of using pDNA rather than mRNA is that the end product has the potential to be contaminated with genomic DNA (gDNA). Also, the isolated pDNA forms three configurations; linear, super-coiled and circular that do not transfect cells with the same efficiency. The mRNA final product is pure, in a single conformation, and is not contaminated with gDNA or pDNA.
• Chart 1 compares steps employed for DNA and mRNA production.
• Chart 2 compares processing of DNA and mRNA in tumor tissue through preparation to vaccination in transfected cells.
• mRNA delivery into the cells skips ahead to immediate translation into the antigenic M-like protein. Not only does transfected DNA have to pass through an additional cellular membrane, but it also has to be transcribed into mRNA for delivery back into the cytosol, which is the starting point for the protein synthesis initiated by the mRNA vaccine.
• mRNA vaccines can be conjugated with compatible immunologic adjuvants or repressors depending on the effect desired.
• Adjuvants such as TriMix, a cocktail of immunostimulatory molecules, can be added to an mRNA-based vaccine eliciting an increased immune response against the encoded immunogen.
• Immunologic repressors can be useful to combat immunosuppressive enzymes of other elements that may hinder the body’s ability to mount a sufficient immune response. These immunosuppressive elements can be silenced by using silencing RNA (siRNA) that can be co-delivered during immunization.
• siRNA silencing RNA
• An additional type of immune repressor that can be administered in conjunction with an mRNA based cancer vaccine is a check-point inhibitor.
• the vaccine can be used not only in conjunction with checkpoint inhibitor therapy but also chemotherapy, radiation therapy, whole cell vaccines, other nucleic acid therapy, natural killer cell therapy or chimeric antigen receptor therapy prior to or concurrently with administration of the RNA vaccine.
• a cancer patient is treated with regimens that alter the tumor microenvironment, including but not limited to, cytokines, anti-fugetaxis agents, chemotactic agents and metronomic doses of chemicals prior to or concurrently with administration of the vaccine.
• regimens that alter the tumor microenvironment including but not limited to, cytokines, anti-fugetaxis agents, chemotactic agents and metronomic doses of chemicals prior to or concurrently with administration of the vaccine.
• Chart 3 compares DNA and mRNA processing in cells from cell entry to translation.
• mRNA encoding emmL can be produced using an in vitro transcription reaction. Several modifications to the resultant mRNA can be made in this reaction to improve mRNA and EmmL protein stability and translation efficiency, and to reduce mRNA immunogenicity.
• a modified nucleic acid can be attached to the 5’ end of emmL mRNA such as, but not limited to, anti-reverse Cap Analog [ARC A, Pl-(5'- (3'-0-methyl)-7-methyl-guanosyl) P3-(5'-(guanosyl))triphosphate)], Nl-methyl-guanosine, T fluoro-guanosine, 7-deaza-guanosine, inosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine and 2-azido-guanosine.
• anti-reverse Cap Analog [ARC A, Pl-(5'- (3'-0-methyl)-7-methyl-guanosyl) P3-(5'-(guanosyl))triphosphate)]
• Nl-methyl-guanosine T fluoro-guanosine, 7-deaza-guanosine, inosine, 8-o
• a poly(A) tail approximately 50-200 adenosine monophosphates in length can be attached to the 3’ end of emmL mRNA or both 5’ modified nucleotide cap and poly(A) tail can be added to emmL mRNA.
• emmL mRNA can be synthesized with ribonucleotide analogs. Chemical modifications can be made at this stage to further improve translation efficiency and stability. Examples include; 5-methyl-cytidine-5’ -triphosphate, pseudouridine-5'-triphosphate, 2- thiouridine-5'triphosphate, and Nl-methylpseudouridine-5'-triphosphate.
• EmmL protein will be produced at high levels.
• emmL mRNA produced following in vitro transcription can be injected directly into a tissue or tumor.
• lipids or polymers can be used to protect RNA from degradation, enhance uptake by cells and improve delivery to the translation machinery in the cytoplasm.
• emmL mRNA is complexed with a liposome prepared from a lipophilic material such as cholesterol and synthetic phospholipids.
• emmL mRNA is complexed with a liposome prepared from a lipophilic material such as cholesterol and natural phospholipids.
• emmL mRNA is complexed with a cationic lipid such as, but not limited to, N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP).
• DOTAP N-[l-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate
• emmL mRNA is complexed with a zwitterionic lipid such as, but not limited to, 3-[(3-Cholamidopropyl)dimethylammonio]-l-Propanesulfonate (CHAPS).
• emmL mRNA is complexed with a PEGylated lipid such as, but not limited to, N-(Carbonyl-methoxypolyethyleneglycol_2000)-l,2-distearoyl-sn-glycero-3 phosphoethanolamine (DSPE-PEG).
• emmL mRNA is complexed with a mixture of cationic, zwitterionic and PEGylated lipids.
• emmL mRNA is complexed with protamine.
• emmL mRNA is complexed with a liposome prepared from a specific material such as the Hemagglutinating virus of Japan, which has been used to prepare mRNA vaccine for the treatment of melanoma [26]
• emmL mRNA can be complexed with a polymer that is rationally designed with multiple materials that mimic viral components to transfect specific cells with high efficiency. These would include, but are not limited to membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand.
• complexing components can be combined and formulated into a single nanoparticle.
• emmL mRNA or emmL mRNA complexes can be combined with interfering RNAs or interfering RNA complexes directed against immune checkpoint molecules such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD- Ll), cytotoxic T-lymphocyte associated protein 4 (CTLA-4) or T-cell immunoreceptor with Ig and ITIM domains (TIGIT).
• interfering RNAs or interfering RNA complexes directed against immune checkpoint molecules such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD- Ll), cytotoxic T-lymphocyte associated protein 4 (CTLA-4) or T-cell immunoreceptor with Ig and ITIM domains (TIGIT).
• PD-1 programmed cell death protein 1
• PD- Ll programmed death-ligand 1
• CTLA-4 cytotoxic T-lymphocyte associated protein 4
• T-cell immunoreceptor with Ig and ITIM domains T-cell immunore
• the emmL coding region can be combined with viral RNA replication genes to form a linear RNA molecule capable of self-replication.
• This linear RNA molecule can then be formulated with lipophilic compounds and lipids to form liposomes capable of transfecting cells with self-replicating mRNA
• emmL mRNA or emmL mRNA formulated with lipids, protamine or in liposomes is complexed with a biodegradable polymer.
• a biodegradable polymer is polycaprolactone, which has been approved by the Food and Drug Administration for use as a drug delivery device.
• the advantage of biodegradable polymer complexing is that mRNA or mRNA complexes can be delivered over a long-time frame as the polymer degrades. This sustained delivery can enhance the effectiveness of a vaccine.
• emmL mRNA or emmL mRNA complexes can be formulated in a biodegradable polymer containing tissue- or tumor-specific factors that enhance the efficacy of the emmL mRNA vaccine.
• a biodegradable polymer containing tissue- or tumor-specific factors that enhance the efficacy of the emmL mRNA vaccine.
• An example can be an emmL mRNA vaccine formulated with a biopolymer containing factors that inhibit angiogenesis or vasculogenesis, thereby providing a synergistic anti-tumor effect.
• emmL mRNA or emmL mRNA complexes can be formulated in a biodegradable polymer containing factors that reduce the expression of immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 or TIGIT.
• immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 or TIGIT.
• this can include existing or novel pharmaceutical reagents such as small molecules or antibodies that reduce the expression of PD-1, thereby providing a synergistic anti-tumor effect.
• emmL mRNA or emmL mRNA complexes can be formulated in a biodegradable polymer containing interfering RNAs or interfering RNA complexes for factors that reduce the expression of immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 or TIGIT.
• interfering RNAs or interfering RNA complexes for factors that reduce the expression of immune checkpoint molecules such as PD-1, PD-L1, CTLA-4 or TIGIT.
• emmL mRNA complexes and short interfering RNA (siRNA) complexes are non-limiting example.
• emmL mRNA or emmL mRNA complexes can be combined with an adjuvant such as synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(LC)].
• an adjuvant such as synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(LC)].
• Proximal elements are close to the emmL coding region and are added during polymerase chain reaction (PCR) amplification of emmL from pAc Iemm55.
• Distal elements are distant from the emmL coding region and are of general utility for enhancing expression of protein from an mRNA.
• Proximal elements include the introduction of a translation initiation sequence optimized for mammalian cell expression to the N-terminal end of the emmL coding region. This sequence has been determined to be RYMRMVATGGC, where R is A or G, Y is C or T, M is A or C and V is A, C or G (SEQ ID NO: 4).
• the optimal translation initiation sequence ATAGCCATGGC (SEQ ID NO: 5), replaces the native start codon of emmL.
• oligonucleotide PCR primers are synthesized such that there is an equimolar concentration of each nucleotide at the degenerate positions (R, Y, M and V in SEQ ID NO: 4) and the final oligonucleotide synthesis product contains all possible primer sequences.
• the optimal translation initiation sequence is determined empirically by comparing the level of EmmL protein expression in assays such as western blots after the emmL gene has been sub cloned into a mammalian expression plasmid DNA vector.
• proximal design element that can be used to modify the C-terminal end of the emmL coding region is the addition of stop codons added after the native stop codon.
• Many eukaryotic expression plasmid vectors contain the DNA sequence of all three stop codon variants, TAG, TAA and TGA, immediately after the last codon of the expressed gene. Since the DNA sequence of the native emmL stop codon is TAG, SEQ ID NO: 6. can be included as an added proximal design element.
• Another proximal design element is the addition of restriction endonuclease recognition sites that can be added to the N- and C-terminal ends of the emmL coding region to facilitate its insertion into plasmid cloning vectors.
• the choice of restriction endonuclease recognition site is made based on the destination plasmid.
• Some examples of restriction endonucleases that could be used are Sacl, Notl, Bglll, EcoRV and Spel.
• Sacl and Spel sites are added to the N- and C-terminal ends of the emmL coding region, as they do not cut within the emmL coding region and share reaction conditions, so they can be used simultaneously to prepare the emmL coding region PCR amplimer for insertion into the destination plasmid.
• Design elements that are distal to the emmL coding region are added to a plasmid cloning vector such that any variation of the emmL coding region, or other gene that is adapted with the proximal design elements, can be inserted. These distal elements can be engineered to create an mRNA expression vector capable of driving high-level mRNA and protein expression of emmL or any other mammalian mRNA.
• One distal design element is the DNA sequence for a bacteriophage RNA polymerase promoter region.
• bacteriophage RNA polymerases are T7, T3 and SP6.
• the promoter for T7 RNA polymerase (SEQ ID NO: 8) is the first of several distal elements added in such a way that it is ultimately upstream, with respect to the action of T7 RNA polymerase, from the emmL coding region.
• UTRs that support high level protein expression are unstructured, lack negative regulatory sequences and may contain the binding sites for microRNAs that serve to further refine the gene expression pattern.
• Both UTRs of genes that are highly expressed in mammalian cells in general, or UTRs from genes with an expression pattern matching the tissue where mRNA vaccine expression is desired, can be used to optimize delivery of a gene product such as EmmL protein.
• UTRs from the Xenopus laevis beta globin gene have been used extensively to mediate high levels of translation in a wide range of eukaryotic cells, including in the design of RNAs used for mammalian immune therapy
• tissue-specific UTRs are the tryptophan hydroxylase (TPH) isoforms, which drive differential expression in the pineal gland and brain stem.
• TPH tryptophan hydroxylase
• the Xenopus laevis beta globin gene 5’ UTR can be positioned upstream, with respect to RNA polymerase activity, from the emmL coding region.
• SEQ ID NO: 9 is one non-limiting example of a Xenopus laevis beta globin gene 5’ untranslated region.
• the Xenopus laevis beta globin gene 3’ UTR can be positioned downstream, with respect to RNA polymerase activity, from the emmL coding region.
• SEQ ID NO: 10 is one non-limiting example of a Xenopus laevis beta globin gene 3’ untranslated region.
• both the Xenopus laevis beta globin gene 5’ and 3’ UTRs can be positioned to flank the emmL coding region.
• 5’ and 3’ UTRs are selected based on the type of neoplastic cell targeted by the vaccine.
• the 5’ and 3’ UTRs of genes expressed highly in melanoma cells such as tyrosinase (TYR), melanogenesis associated transcription factor (MITF), melanocortin receptor 1(MC1R), telomerase (TERT), cyclooxygenase 2 (COX2), C-X-C motif chemokine receptor 4 (CXCR4) and baculoviral IAP repeat containing 5 genes (BIRC5), can be used to provide high levels of cell-specific expression to an emmL- based vaccine when used to treat melanoma.
• other genetic elements not located within the primary gene transcript that confer desired expression level and specificity can be included before, after or within the 5’ and 3’ UTRs to further refine vaccine expression.
• Another distal design element is a synthetic DNA sequence that provides restriction endonuclease recognition sites that can be used to insert the emmL coding region into the other flanking distal design elements.
• the restriction endonuclease recognition sites for Sacl, Notl, Bglll, EcoRV and Spel are used (SEQ ID NO: 7).
• Another distal design element is a synthetic DNA sequence that provides restriction endonuclease recognition sites to cut the destination plasmid immediately after the emmL coding region to produce a linear plasmid DNA molecule. Linearization provides effective termination for RNA polymerase activity, which increases the production of uniform length mRNA transcripts.
• An example of a sequence is SEQ ID NO: 11, which contains restriction endonuclease recognition sites for BamHI, EcoRI and Xbal.
• BamHI can be used to linearize a plasmid containing the emmL coding region as it does not cut within the emmL coding region and leaves a 5’ nucleotide overhang, which may allow for higher efficiency transcription than a 3’ nucleotide overhang.
• a double stranded DNA molecule containing the abovementioned distal design elements is synthesized by use of overlapping complementary synthetic single stranded oligonucleotide molecules. These molecules are annealed and gaps remaining are filled in with a DNA polymerase such as Taq polymerase.
• two complementary synthetic single stranded oligonucleotide molecules can be synthesized to encompass all desired distal design elements. These oligonucleotide molecules are then annealed.
• the resultant blunt-ended synthetic DNA molecule can be inserted into a PCR cloning vector such as the Invitrogen pCR ® II-TOPO plasmid by adding a template non-specific adenosine. This is accomplished by incubating the blunt-ended synthetic DNA molecule with Taq polymerase and deoxyadenosine triphosphate at approximately 200 mM final concentration at 72°C for 10 minutes.
• a plasmid containing the abovementioned distal design elements is named pT7XLUTR.
• a map of the synthetic DNA molecule inserted into pCR ® II- TOPO is shown as FIG. 11 and the nucleotide sequence is shown as SEQ ID NO: 12. This sequence contains the abovementioned distal design elements, but does not represent the totality or full extent of elements that could be added to support the desired expression level or specificity.
• pT7XLUTR can be used to transform E. coli.
• Bacterial transformed with pT7XLUTR can be selected on an agar plates containing an antibiotic that matches the PCR cloning vector, such as agar plates containing Luria Bertani broth supplemented with 50-100 pg/mL kanamycin or carbenicillin.
• the bacterial culture can be expanded by growth in a liquid antibiotic selective media, such as Luria Bertani broth supplemented with 50-100 pg/mL kanamycin or carbenicillin, and plasmid DNA prepared using methods known to a person practiced in the art.
• RNAs incorporating several of the described design elements were found to increase expression of Emm55.
• the sequences are shown in SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 16.
• Example 1 Autologous mRNA Vaccine for Canine Lymphoma
• a 75 lb. male neutered Rhodesian Ridgeback presents to his veterinarian with swollen mandibular and inguinal lymph nodes. The patient has a fine needle aspirate performed on one of the enlarged nodes. Upon review by the pathologist he is diagnosed with low grade diffuse lymphoma.
• the patient’s owner elects to pursue immunotherapy treatment instead of chemotherapy and steroids, due to the minimal side effects reported in immunotherapy treatments.
• the veterinarian excises the right mandibular lymph node while the patient is under general anesthesia.
• the tissue sample is shipped overnight for laboratory processing. [00126] Upon receiving the tissue sample at the lab the following is performed: 1) the travel medium is checked for any bacterial contamination, 2) the tissue dimensions are measured, 3) the intact lymph node is aspirated repeatedly using several boluses of wash medium to release the tumor cells, and 4) the aspirated cells are collected and counted.
• the 8 vaccine doses are given every 7 days (+/- 1 day) for 4 weeks and then once a month for 4 months. Prior to the first dose a blood sample is taken. Subsequent blood samples are taken preceding the 5 th vaccine, 8 th vaccine and 8 weeks after the last vaccine. The blood samples are processed for peripheral blood & plasma and preserved at the lab. They are later used for evaluation of anti-tumor immune response.
• lymph node size is monitored along with his overall quality of life. Overall disease state is assessed by tumor burden reduction and anti-tumor immune response. Tumor burden is evaluated through measurements performed on each of the lymph nodes throughout the course of treatment. Anti-tumor immune response is measured using standard enzyme-linked immunosorbent assay (ELISA) to assess antibody levels and flow cytometry to assess cytotoxic T-cell (CTL) response.
• ELISA enzyme-linked immunosorbent assay
• lymph node size increases and later decreases as the course continues. This observation is probably due to infiltration of immune cells into the tumor site, in this case, the lymph nodes.
• the ELISA and flow cytometry results show an increase in antibody production and CTLs after the fourth vaccine that then persists after the completion of the series of vaccines.
• Example 2 Direct mRNA Vaccine for Equine Melanoma
• Three vaccine doses are prepared containing 100 pg mRNA in 100 pL sterile nuclease free H 2 0.
• the three doses and three needle-free injection devices (J-Tip) are shipped to the veterinarian.
• Three of the patient’s lesions are chosen to receive the treatment course, a total of 300 pg mRNA per time point. Every two weeks three more doses are shipped to the veterinary clinic as previously done and each dose is administered to the same three lesions using the J-Tip device.
• the patient receives a total of six vaccine doses per lesion.
• Blood samples are collected prior to initiation of the vaccine series, prior to the 5 th vaccine dose and two weeks after the series is completed. The blood samples are processed for peripheral blood & plasma and preserved. They are later used for evaluation of anti-tumor immune response.
• the melanoma lesions will initially increase in size followed by a decrease as the vaccine series progresses.
• the ELISA and FACS results show an increase in antibody production and CTLs after the second vaccine that will persist after the completion of the series of vaccines.
• Example 3 Overview of methods for emmL mRNA creation [00138] Methods Overview:
• a plasmid backbone including dual prokaryote and eukaryote promoters, untranslated 3’ and 5’ regions, and a selective marker was used.
• a vector of this type; for example, pSFCMVT7 has multiple features that aid in the production and stabilization of the mRNA encoding an antigenic M-like protein, such as Emm55. Both the vector pSFCMVT7 and the insert containing plasmid pAc /emmL were cut using restriction enzymes Sad and AcoRV. Refer to FIGs 1 and 2 for plasmid maps.
• mRNA production plasmid pSFCMVT7 lemmL was created, it was transformed, i.e., transfected, into competent bacteria that produce sufficient DNA that was isolated and will be used for in vitro mRNA synthesis.
• Invitrogen s Stbl3 E. coli is an example of the type of bacteria that can be used for transfection. Transformation was induced by heat shocking the bacteria to open up small orifices in the cell membranes allowing the plasmid to enter the cell and ultimately the nucleus.
• the bacteria transfected with the plasmid were placed onto appropriate growth medium containing a selective antibiotic. In the case of pSFCMVT7 this is a kanamycin. If the bacteria are correctly transformed with the plasmid they will produce a protein that will hinder the anti -bacterial properties of the kanamycin and allow the kanamycin-resistant bacteria to selectively grow on the medium.
• the isolated DNA contains the template DNA for mRNA production.
• the plasmid In order for the transcription reaction to occur, the plasmid must be linearized. It is important that the linearization occur down-stream from the open reading frame gene of interest.
• the message is created through an in vitro transcription reaction. This reaction simulates the transcription of mRNA in the cell, including the capping of the 5’ end and addition of a poly(A) tail for increased stabilization.
• mRNA Purification [00158] Once the message has been transcribed into mRNA, the residual DNA template is degraded so that a pure mRNA product can be used to transfect into autologous cells, allogeneic cells or intratum orally. Once inside the cells the mRNA will produce and display the M-like protein on the cell surface for immune activation.
• mRNA can be delivered into the cancer cells is by the method of electroporation. This method utilizes a weak electrical current that causes the cellular membrane to open up small pores that then allow the mRNA to move through the membrane and into the cytoplasm.
• Table 1 shows the procedure for rapid digestion of pDNA.
• Example 5 DNA fragment separation with gel electrophoresis.
• Table 2 shows the procedure for DNA fragment separation.
• Table 3 shows the procedure for Extraction and DNA Isolation.
• Example 7 Vector and Insert Ligation.
• Table 4 shows the procedure for vector insert and ligation.
• Table 5 shows the procedure for transformation of E. coli.
• the Table 6 shows the procedure for growth and expansion of the bacterial culture.
• Table 7 shows the procedure for plasmid isolation and purification.
• Table 8 shows the procedure for preparation of template DNA and plasmid linearization.
• Table 9 shows the procedure for transcribing mRNA TABLE 9
• Table 10 shows the procedure for purifying the mRNA
• Table 11 shows the procedure for transfection of mammalian cancer cells with emmL mRNA.
• Example 15 Cloning steps for DNA pSFCMVT7 lemmL.
• FIG. 5 shows the procedure for creating a recombinant DNA vector to produce mRNA encoding bacterial antigen.
• Example 16 Direct binding of antibody to M-like protein.
• the Western blot shown in FIG. 6. demonstrates the specificity of the anti -M-like protein antibody to isolated M-like protein, specifically Emm55.
• Proteins were separated by SDS-PAGE (10%) using 130mM b-ME in the loading buffer. Samples were boiled for 3 minutes at 100°C and spun at 13,000 x g for 2 minutes at room temperature. The blots (far left) were probed with primary antibody (a-M-like Protein) for 1.5 hours at room temperature in 5% milk. The primary antibody dilution was 1:500. The secondary antibody (goat a-mouse conjugated HRP) was at a dilution of 1 : 5000. The null blot (second from left) shows the non-specific binding of secondary antibody.
• Chemiluminescence was used to visualize the protein on the nitrocellulose blots (exposure: 10 minutes).
• Example 17 Fluorescence microscope images and chart demonstrating the increased expression seen with mRNA as compared with DNA. Results were compared from an experiment in which RNA and DNA were transfected into mammalian cells and assayed for protein expression. The results show that RNA at equivalent transfection quantity produced five times the amount of expression. (See FIG. 7).
• Example 18 Synthesized emmL mRNA, untailed and tailed.
• RNA samples and 100 ng ladder were prepared by diluting the total quantity into a 2.5 pL volume using DEPC treated water. An equivalent volume of formaldehyde sample buffer was added to each sample. The samples were mixed and then incubated at 65°C for 15 minutes followed by a 1 minute incubation on ice. Samples were loaded into a 1.2% RNA gel cassette and then run at 225 volts for 8 minutes. The gel was incubated at room temperature for 10 minutes and then visualized using the FlashGel camera. mRNA sizes are determined by the RNA Millennium Marker.
• Chart 4 displays the results from an experiment in which RNA ⁇ emmL mRNA) and DNA (pSFCMVT17/ewwZ) were transfected into mammalian cells, stained with a-M-like protein, and assayed using flow cytometric analysis. The results show that the RNA- transfected cells produce an equivalent signal to the DNA transfected cells, i.e. 9%.
• Chart 5 shows the results from an experiment in which mice were transplanted with melanoma tumor cells and subsequently injected with either emmL mRNA (treatment) or sterile water (control).
• the injection regimen began 10 days after tumor implantation.
• the regimen consisted of three injections, of either treatment or control, administered every seven days. All five mice in the experiment lived past injection #2. At this time, two out of three treatment mice had smaller tumors than the control mice. Three of the five mice survived past injection #3, at which point the two remaining treatment mice tumors were still smaller than the remaining control mouse tumor.
• FIG. 10 shows results from an experiment in which blood samples from mice, pre and post vaccination with emmL mRNA, were tested for the presence of antibodies that react to emmL protein.
• the Western blot images indicate that the blood samples taken post vaccination have increased binding of antibodies from the pre-vaccination sample.
• a PCR with high fidelity Taq DNA polymerase is used to amplify the emmL coding region from the pAc Iemm55 plasmid.
• this amplimer is designed with proximal elements that include restriction endonuclease sites compatible with the pSF- CMV T7 vector to minimize potentially inhibitory elements within the 5’ and 3’ UTRs of pSFCMVT7/ewwZ.
• the proximal design elements include restriction endonuclease recognition sites for Ncol and Xhol, an optimal translation initiation sequence (SEQ ID NO: 4 or 5) and two additional stop codons (SEQ ID NO: 6) downstream of the emmL coding region, with respect to the activity of T7 RNA polymerase.
• the resultant amplimer can be inserted into a PCR cloning vector such as pCR ® II-TOPO.
• the resultant plasmid can be used to transform E.
• Bacterial cultures containing the resultant plasmid can expanded by growth in a liquid antibiotic selective media, such as Luria Bertani broth supplemented with 50-100 pg/mL kanamycin or carbenicillin in the case of pCR ® II-TOPO. Plasmid DNA can be prepared from these bacterial cultures using methods known to a person practiced in the art.
• restriction endonucleases Ncol and Xhol can then be used to excise the emmL coding region flanked by proximal design elements from pCR ® II-TOPO plasmid DNA.
• the emmL coding region flanked by proximal design elements DNA fragment can then be inserted into the pSF-CMV_T7 vector, which has been digested with Ncol and Xhol.
• the resultant plasmid, pSF lemmL can be used as a template for in vitro mRNA synthesis following linearization with the restriction endonuclease XhoT
• SEQ ID NOs: 13 and 14 The resultant mRNA and predicted amino acid sequences are shown as SEQ ID NOs: 13 and 14, respectively.
• High fidelity Taq DNA polymerase is used to amplify the emmL coding region from the pAc !emm55 plasmid with proximal design elements that include restriction endonuclease sites compatible with the polylinker region of pT7XLUTR.
• this can also include an optimal translation initiation sequence (SEQ ID NO: 4 or 5) and two additional stop codons (SEQ ID NO: 6).
• SEQ ID NO: 6 The full sequence of this embodiment is shown in SEQ ID 15.
• the resulting amplimer can be inserted into a PCR cloning vector such as pCR ® II-TOPO.
• the resultant plasmid can be digested with restriction endonucleases Sacl and Spel to excise the emmL coding region with proximal design elements from pCR ® II- TOPO.
• the emmL coding region with proximal design elements can then be ligated into the pT7XLUTR plasmid vector that has been digested with Sacl and Spel to generate pT7XLUTR/ewwZ.
• the resultant pT7XLUTRA/w??/. plasmid can be used to transform E. coli.
• the resultant pT7XLUTR/ewwZ plasmid can be used as a template for in vitro mRNA synthesis following linearization with the restriction endonuclease BamHI.
• the resultant mRNA and predicted amino acid sequences are shown as SEQ ID NOs: 15 and 14, respectively.
• Example 25 High fidelity Taq DNA polymerase is used to amplify the emmL coding region from the pAdemm55 plasmid with minimal 5’ and 3’ UTRs.
• the T7 RNA polymerase promoter sequence (SEQ. ID NO 8) is added as a proximal design element along with an optimal translation initiation sequence (SEQ ID NO: 4 or 5), two additional stop codons (SEQ ID NO: 6) and an Xhol restriction endonuclease site.
• the resultant PCR product can be inserted into a PCR cloning vector such as pCR ® II-TOPO.
• a PCR cloning vector such as pCR ® II-TOPO.
• the resultant plasmid, pT71 emmL can be used as a template for in vitro mRNA synthesis following linearization with the restriction endonuclease Xhol.
• the resultant mRNA and predicted amino acid sequences are shown as SEQ ID NOs: 16 and 14, respectively.
• mRNA encoding emmL can be produced using an in vitro transcription reaction. Several modifications to the resultant mRNA can be made in this reaction to improve mRNA and EmmL protein stability and translation efficiency, and to reduce mRNA immunogenicity.
• a modified nucleic acid can be attached to the 5’ end of emmL mRNA such as, but not limited to, anti-reverse Cap Analog [ARC A, Pl-(5'- (3'-0-methyl)-7-methyl-guanosyl) P3-(5'-(guanosyl))triphosphate)], Nl-methyl-guanosine, T fluoro-guanosine, 7-deaza-guanosine, inosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine and 2-azido-guanosine.
• anti-reverse Cap Analog [ARC A, Pl-(5'- (3'-0-methyl)-7-methyl-guanosyl) P3-(5'-(guanosyl))triphosphate)]
• Nl-methyl-guanosine T fluoro-guanosine, 7-deaza-guanosine, inosine, 8-o
• a polyadenylated [poly(A)] tail approximately 50-200 adenosine monophosphates in length can be attached to the 3’ end of emmL mRNA or both 5’ modified nucleotide cap and poly(A) tail can be added to emmL mRNA.
• emmL mRNA can be synthesized with ribonucleotide analogs. Chemical modifications can be made at this stage to further improve translation efficiency and stability. Examples include; 5-methyl-cytidine-5’ -triphosphate, pseudouridine-5'-triphosphate, 2- thiouridine-5'triphosphate, and Nl-methylpseudouridine-5'-triphosphate.
• SEQ ID NO: 4 Degenerate DNA sequence of an optimal translation initiation sequence.
• R is A or G
• Y is C or T
• M is A or C
• V is A, C or G.
• SEQ ID NO: 5 DNA sequence of an optimal translation initiation sequence.
• SEQ ID NO: 6 DNA sequence containing 2 stop codons. [00219] TAATGA
• SEQ ID NO: 7 DNA sequence of the restriction endonuclease recognition sites for Sacl, Notl, Bglll, EcoRV and Spel.
• SEQ ID NO: 8 DNA sequence of the T7 RNA polymerase promoter.
• SEQ ID NO: 10 74 ribonucleotides of the Xenopus laevis beta globin gene 3’ untranslated region.
• SEQ ID NO: 11 DNA sequence of the restriction endonuclease recognition sites for BamHI, EcoRI and Xbal.
• SEQ ID NO: 12 DNA sequence of the synthetic double stranded DNA molecule used to prepare pT7XLUTR. T7 RNA polymerase, restriction endonuclease recognition sites for Sacl, Notl, Bglll, EcoRV, Spel, as well as BamHI, EcoRI and Xbal are underlined.
• SEQ ID NO: 13 (RNA sequence of example 23). Synthetic mRNA sequence following in vitro mRNA synthesis of emmL from a plasmid template, written from 5’ to 3’ with respect to the mRNA molecule. An anti -reverse cap analog (ARC A) is added to the 5’ end of the transcript and approximately 50-200 adenosine monophosphates ( a 50-200 ) are added to the 3’ end of the transcript during in vitro transcription. 5’ lowercase letters are the 5’ untranslated region (UTR) from the pSF-T7_CMV plasmid vector.
• UTR untranslated region
• the guanine (g) immediately after the ARCA cap is the +1 ribonucleotide produced by T7 RNA polymerase.
• Bold indicates ribonucleotides added to make the 5’ Ncol restriction endonuclease site, which is underlined, and an optimal translation initiation sequence.
• Uppercase letters indicate the coding sequence of emmL.
• bold indicates two additional stop codons.
• the 3’ Xhol restriction endonuclease site is underlined.
• SEQ ID NO: 14 (Predicted protein sequence of examples 23-25). One letter predicted amino acid sequence of emmL transcripts produced in examples 2-4. * indicates stop codon.
• SEQ ID NO: 15 (RNA sequence of example 24). Synthetic mRNA sequence following in vitro mRNA synthesis of emmL from the pT7XLURT plasmid template linearized with restriction endonuclease BamHI, written from 5’ to 3’ with respect to the mRNA molecule. An anti-reverse cap analog (ARC A) is added to the 5’ end of the transcript and approximately 50-200 adenosine monophosphates (a 50-200 ) are added to the 3’ end of the transcript during in vitro transcription. The guanine (g) immediately after the ARCA cap is the +1 ribonucleotide produced by T7 RNA polymerase.
• ARC A anti-reverse cap analog
• a 50-200 adenosine monophosphates
• 5’ lowercase letters are from the 5’ untranslated region of the Xenopus laevis beta globin gene. Bold indicates ribonucleotides added to make an optimal translation initiation sequence.
• a Sacl restriction endonuclease site 5’ of the emmL coding region is underlined. Uppercase letters indicate the coding sequence of emmL.
• Lowercase letters 3’ of the coding region are the 3’ untranslated region of the Xenopus laevis beta globin gene. At the 3’ end of the mRNA sequence, bold indicates two additional stop codons. Spel and BamHI restriction endonuclease sites are underlined.
• SEQ ID NO: 16 (RNA sequence of example 24). Synthetic mRNA sequence following in vitro mRNA synthesis of emmL from a plasmid template, written from 5’ to 3’ with respect to the mRNA molecule. An anti -reverse cap analog (ARC A) is added to the 5’ end of the transcript and approximately 50-200 adenosine monophosphates (a 50 200 )are added to the 3’ end of the transcript during in vitro transcription. The guanine (g) immediately after the ARCA cap is the +1 ribonucleotide produced by T7 RNA polymerase. Bold indicates ribonucleotides added to make an optimal translation initiation sequence.
• ARC A anti -reverse cap analog
• a 50 200 adenosine monophosphates
• Codon optimization algorithms were also used to synthesize emm55 mRNAs. Emm55 expression was measured and compared to determine relative increase protein synthesis and stability in mammalian cells. Each mRNA was synthesized in vitro using wild type uridine (WT) and Nl-methylpseudouridine (Nl) was shown to improve protein expression.
• WT wild type uridine
• Nl Nl-methylpseudouridine
• the final nucleotide sequence for each mRNA is shown as SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
• SEQ ID NO: 17 The final nucleotide sequence for each mRNA is shown as SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
• Bbsl and BspQI restriction endonuclease sites were removed by base substitution from emm55JCat and emm55MostFreq to facilitate cloning into the TriLink® plasmid vector used to synthesize mRNA in vitro.
• mRNAs were received with each tube of mRNA supplied by TriLink® Biotechnologies and added as a stock item so that each tube could be tracked independently. mRNAs were stored at -80°C. Quality control testing was carried out to verify purity and intactness using RD 3-75.1, except that mRNAs were aliquoted by TriLink® Biotechnologies rather than as specified in the SOP (standard operating procedure).
• mRNAs were transiently transfected into the attachment enhanced HEK subline HEK293T-AE and B16-F10 cells using RD 3-79.1 (2.5 pg mRN A/well). Cells were transfected with pAc !emm55 using RD 3-34.2 (4 pg pDNA/well). Two to 48 hours post transfection, cells were lysed in RIPA buffer using RD 3-83.1 and the protein concentrations of the lysates was determined by BCA assay using RD 3-14.2.
• EGFP mRNA and pDNA were used as transfection positive controls and Emm55 expression negative controls. Initially, lysates were resolved using denaturing SDS-PAGE, western blotted, stained with chicken anti-Emm55 and detected using chemiluminescence using RD 3-74.1. The band corresponding in size to Emm55 consistently overwhelmed the peroxidase detection reaction, resulting in bands that could not be quantified. No suitable dilution of chicken anti-Emm55 or goat anti -chicken-horseradish peroxidase could be identified to increase the high end of the detection range.
• Emm55 protein detection was carried out using the ProteinSimple WesTM capillary electrophoresis system using chicken anti-Emm55. This system resolves protein and protein/antibody complexes in a reducing SDS-PAGE capillary tube to provide antibody binding quantification like western blot data. These assays were carried out using manufacturer-supplied protocol.
• the WesTM assay measured the level of Emm55 in cell lysate normalized to the level of ERK1, a serine/threonine kinase that is regulated by phosphorylation rather than protein level, and thus makes a suitable protein for data normalization within a cell or tissue type.
• This approach has an advantage over internal total protein normalization, as it can be multiplexed with Emm55 detection, providing a sample loading control and cutting in half the number of sample assays that need to be performed.
• a standard curve was constructed with 10 ng to 30 pg rEmm55 in 1.5 pg untransfected HEK293T-AE lysate.
• Antibody dilutions for the WesTM assay were; chicken anti-Emm55 at 1 : 100 and goat anti -chi cken-HRP at 1 : 100.
• the WesTM assay assembly instructions refer to input protein concentration rather than mass.
• Experiments using the WesTM were carried out using cell lysates at 0.1, 0.25 and 0.5 pg/pL. While 0.5 pg/pL gave results within the WesTM dynamic range with B16-F10 cell lysates, 0.25 and 0.5 pg/pL HEK293T lysates generated expression levels that exceeded the detection capacity at peak expression time points.
• a concentration of 0.1 pg/pL lysate was found to be optimal throughout the time course, although lower concentrations could be used to provide additional dynamic range when high level expression is anticipated. However, this lower input concentration may miss low but biologically meaningful levels of expression, especially at early and late time points.
• a codon adaptiveness index (CAI) was calculated using the JCat algorithm for each codon of emm55. This algorithm returns a score of 1.0 if the mRNA sequence is perfectly optimized for translation in human cells.
• FIG. 12 shows the relative adaptiveness of emm55 before and after optimization with JCat. A relative adaptiveness plot of emm55 is shown in FIGs 12A and 12B.
• Results showed that codon modified emm55 mRNA drives robust expression of Emm55 polypeptide following transient transfection of human HE293T cells and murine B16-F10 melanoma cells in vitro.
• a comparison of protein levels to levels of transfection with pAc/emm55 (the DNA) is not quantitative since a single copy of pDNA gives rise to many copies of mRNA but did show that WT and Nl-modified emm55JCat expressed significantly higher levels of Emm55 in HEK293 cells between 4-and 12-hours post transfection. Peak Emm55 expression from emm55JCat mRNA was approximately 12 hours post-transfection and was detected at 48 hours.

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