Non-stochastic generation of genetic vaccines

A method for obtaining an immunomodulatory polynucleotide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, or encodes a polypeptide that has an optimized modulatory effect on an immune response as compared to the response prior to optimization, the method comprising:

• a) creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set;
• and
• b) screening the library to identify an optimized non-stochastically generated progeny polynucleotide that has, or encodes a polypeptide that has, a modulatory effect on an immune response induced by a vector;
• wherein the optimized non-stochastically generated polynucleotide or the polypeptide encoded by the non-stochastically generated polynucleotide exhibits an enhanced ability to modulate an immune response compared to a parental polynucleotide from which the library was created;
• and
• whereby optimization is achieved using one or more directed evolution methods in any combination, permutation, and iterative manner.

The method of 33, wherein the cytokine is interferon-α and the screening is performed by:

• i) expressing the non-stochastically generated polynucleotides so that the encoded polypeptides are produced as fusions with a protein displayed on the surface of a replicable genetic package;
• ii) contacting the replicable genetic packages with a plurality of B cells;
• and
• iii) identifying phage library members that are capable of inhibiting proliferation of the B cells.

A method for obtaining an optimized polynucleotide that encodes an accessory molecule that improves the transport or presentation of antigens by a cell, the method comprising:

• a) 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;
• and
• b) screening the library to identify an optimized non-stochastically generated progeny polynucleotide that encodes a recombinant molecule that confers upon a cell an increased or decreased ability to transport or present an antigen on a surface of the cell compared to an accessory molecule encoded by template polynucleotides not subjected to the non-stochastic reassembly;
• whereby application of the method can thus be used to generate an optimized molecule for human recipients &/or animal recipients, including animals that are farmed or raised by man, animals that are not farmed or raised by man, domesticated animals, and non-domesticated animals;
• whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation, and iterative manner;
• whereby these directed evolution methods include the introduction of point mutations by non-stochastic methods, including by “gene site saturation mutagenesis” as described herein;
• and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein;
• including by synthetic ligation polynucleotide reassembly as described herein.

A method for obtaining an immunomodulatory polynucleotide that has, an optimized expression in a recombinant expression host, the method comprising:

• creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set;
• whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation and iterative manner;
• whereby these directed evolution methods include the introduction of mutations by non-stochastic methods, including by “gene site saturation mutagenesis” as described herein;
• and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein;
• including by synthetic ligation polynucleotide reassembly as described herein.

A method for obtaining an immunomodulatory polynucleotide that has an optimized expression in a recombinant expression host, the method comprising:

• a) creating a library of non-stochastically generated progeny polynucleotides from a parental polynucleotide set;
• and
• b) screening a library of non-stochastically generated progeny polynucleotides to identify an optimized non-stochastically generated progeny polynucleotide that has an optimized expression in a recombinant expression host when compared to the expression of a parental polynucleotide from which the library was created;
• whereby optimization can thus be achieved using one or more of the directed evolution methods as described herein in any combination, permutation, and iterative manner;
• whereby these directed evolution methods include the introduction of point mutations by non-stochastic methods, including by “gene site saturation mutagenesis” as described herein;
• and whereby these directed evolution methods also include the introduction mutations by non-stochastic polynucleotide reassembly methods as described herein;
• including by synthetic ligation polynucleotide reassembly as described herein.