Non-stochastic generation of genetic vaccines and enzymes

The method of 30, wherein the library of non-stochastically generated polynucleotides is screened by testing the ability of cytokines encoded by the non-stochastically generated polynucleotides to activate cells which contain a receptor for the cytokine.

The method of 30, wherein the cytokine is interleukin-12 and screening is performed by growing mammalian cells which contain the genetic vaccine vector in a culture medium and detecting whether T cell proliferation or T cell differentiation is induced by contact with the culture medium.

The method of 30, wherein the cytokine is interferon-γ.

A method of providing an optimized non-stochastically generated polynucleotide that has a modulatory effect on an immune response said method comprising non-stochastically reassembling at least two parental template polynucleotides, each of which encodes a molecule that is involved in modulating an immune response, thereby providing a library of non-stochastically generated polynucleotides.

A method of providing an optimized polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell, said method comprising creating a library of non-stochastically generated polynucleotides by subjecting to optimization by non-stochastic directed evolution a parental polynucleotide set in which is encoded all or part of the accessory molecule.

A method of providing an immunomodulatory polynucleotide that has an optimized expression in a recombinant expression host, said method comprising creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set, thereby providing an immunomodulatory polynucleotide.

A method of producing a progeny polynucleotide set by subjecting a double-stranded circular parental polynucleotide molecule to mutagenesis, said method comprising synthesizing by means of a polymerase-catalyzed amplification reaction a first progeny polynucleotide strand comprised of said first primer and a second progeny polynucleotide strand comprised of said second primer, wherein the first progeny polynucleotide strand and the second progeny polynucleotide strand form a double-stranded mutagenized circular polynucleotide product, and wherein at least one of said primers contains a non-stochastic mutagenic cassette with respect to the parental polynucleotide molecule, thereby producing a progeny polynucleotide set.

A method of producing a set of progeny polypeptides, in which a non-stochastic range of single amino acid substitutions is represented at each amino acid position, from a template polypeptide set, said method comprising subjecting a codon-containing template polynucleotide to polymerase-based amplification using a degenerate oligonucleotide for each codon to be mutagenized.