Synthetic, persistent RNA vectors for controlled expression of one or more heterologous polynucleotide sequences, each of the one or more heterologous polynucleotide sequences encoding for a reprogramming factor, are described. The vectors comprise a mechanism for silencing (off) and initiation or resumption (on) control of expression of the one or more reprogramming factors in the cell, tissue, or organ. Methods of using the vectors are also described, for example, to treat the age-related disease or condition, where the methods provide for treatment of the disease or condition, and in some embodiments, with retention of cellular identity.

Disclosed herein are compositions that include antigen-encoding nucleic acid sequences and/or antigen peptides. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines, including vectors and methods for a heterologous prime/boost vaccination strategy.

Methods for treating a cell, tissue, or organ and for treating an age-related disease or condition are provided, where the cell, tissue or organ is contacted with a synthetic, persistent RNA vector comprising one or more heterologous polynucleotide sequences, each of the one or more heterologous polynucleotide sequences encoding for a reprogramming factor. Contacting achieves expression of the one or more reprogramming factors in the cell, tissue, or organ to treat the age-related disease or condition. In an embodiment, the method is used to obtain a rejuvenated cell, tissue, or organ with retention of cellular identity.

The subject matter described herein relates to compositions and methods for cellular rejuvenation, tissue engineering, and regenerative medicine by transient exposure of cells or tissues to synthetic, non-integrative mRNAs encoding reprogramming factors. Reprogramming factor encoding polynucleotides and corresponding polypeptides that trigger less immune response, are more stable, and/or exhibit altered activity than wild-type reprogramming factors are provided. RNA vectors expressing one or more of the improved reprogramming factor polynucleotide sequences are also provided.

Provided are polynucleotides and viral vectors, particularly, alphavirus vectors such as Sindbis viral vectors, which encode multiple, e.g., two or more, epitopes of at least one tumor associated antigen in which each epitope is separated by a processing or enzyme cleavage site. The multiple epitopes of the two or more tumor associated antigens encoded by the described polynucleotides and viral vectors may be the same or different. Methods of treating mammalian subjects having a cancer or tumor expressing the tumor associated antigen epitopes are provided, in which the viral vectors encoding the multiple epitopes, as well as other immunostimulatory or immunomodulatory components, generate an anti-cancer or anti-tumor immune response in which high levels of effector T cells increase the survivability of tumored mammalian subjects and result in epitope spreading, thus providing a further enhancement of the immune response.

RNA encoding an immunogen is co-delivered to non-immune cells as the site of delivery and also to immune cells which infiltrate the site of delivery. The responses of these two cell types to the same delivered RNA lead to two different effects, which interact to produce a strong immune response against the immunogen. The non-immune cells translate the RNA and express the immunogen. Infiltrating immune cells respond to the RNA by expressing type I interferons and pro-inflammatory cytokines which produce a local adjuvant effect which acts on the immunogen-expressing non-immune cells to upregulate major histocompatibility complex expression, thereby increasing presentation of the translated protein to T cells. The effects on the immune and non-immune cells can be achieved by a single delivery of a single RNA e.g., by a single injection.

Disclosed herein are self-replicating RNA molecules encoding prostate neoantigens, vaccines, and method of treating and preventing prostate cancer using the self-replicating RNA molecules and vaccines.

Disclosed herein are compositions that include antigen-encoding nucleic acid sequences and/or antigen peptides. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines, including vectors and methods for a heterologous prime/boost vaccincation strategy.

Modified alphaviruses encoding a SARS-CoV-2 spike protein or antigenic segment of the SARS-CoV-2 spike protein are provided. The modified alphaviruses include replicative defective Sindbis viruses. The modified viruses express or are administered with an immunomodulatory agent that is an agonist antibody or antigenbinding fragment thereof, or a cytokine, or a combination thereof. Pharmaceutical compositions that include the modified alphaviruses and methods of using the modified alphaviruses and compositions that contain them are provided. The compositions are used to stimulate a therapeutic or protective effect against SARS-CoV-2 infection that includes humoral and cell mediated responses.

RNA encoding an immunogen is co-delivered to non-immune cells as the site of delivery and also to immune cells which infiltrate the site of delivery. The responses of these two cell types to the same delivered RNA lead to two different effects, which interact to produce a strong immune response against the immunogen. The non-immune cells translate the RNA and express the immunogen. Infiltrating immune cells respond to the RNA by expressing type I interferons and pro-inflammatory cytokines which produce a local adjuvant effect which acts on the immunogen-expressing non-immune cells to upregulate major histocompatibility complex expression, thereby increasing presentation of the translated protein to T cells. The effects on the immune and non-immune cells can be achieved by a single delivery of a single RNA e.g., by a single injection.