Provided herein are methods and compositions useful in the production of recombinant AAV (rAAV) in insect cells. In some embodiments, methods and compositions include the use of modified Kozak sequences to express AAV VP1 proteins in amounts that are useful for producing infective rAAV particles.

Provided are vaccines, polypeptides and polynucleotides based on mutant CALR and JAK2 sequences, vectors, host cells, viruses, and methods of making and using them. The disclosure also provides methods of inducing an immune response and methods of treating, preventing, reducing a risk of onset or delaying the onset of a clinical condition characterized by an expression of JAK2V617F or CALR exon 9 mutant, or both JAK2V617F and CALR exon 9 mutant, wherein the method comprises a plurality of administrations of any of the compositions comprising polynucleotides, polypeptides or vectors disclosed herein.

Disclosed are methods and constructs for engineering circular RNA. Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence:

a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In another embodiment, the vector can comprise the 5′ spacer sequence, but not the 3′ spacer sequence. In yet another embodiment, the vector can comprise the 3′ spacer sequence, but not the 5′ spacer sequence. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AA V) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AA V) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.

A modular and hierarchical DNA assembly platform for synthetic biology is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can precisely assemble multiple DNA fragments in a single reaction using a standardised assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector. We describe the design and use of MIDAS, and its application in the reconstruction of the metabolic pathway for production of paspaline, a key intermediate in the biosynthesis of a range of indole diterpenesa class of economically important secondary metabolites produced by several species of filamentous fungi.

Recombinant constructs, cells and means for improved production of Adeno-Associated Viruses (AAVs) are described. Also described are methods of using the constructs and cells to produce recombinant AAVs.

The present invention relates to a retroviral particle comprising a protein derived from the Gag polyprotein, an envelope protein, optionally an integrase and at least two encapsidated non-viral RNAs, the encapsidated non-viral RNAs each comprising an RNA sequence of interest bound to an encapsidation sequence, each encapsidation sequence being recognized by a binding domain introduced into the protein derived from the Gag polyprotein and/or into the integrase, and at least one of said sequences of interest of the encapsidated non-viral RNAs comprises a part coding a nuclease.

The present invention relates to a method based on the use of restriction enzyme digestion and ligation via the cleavage sites, thereby to prepare two or more standardized expression cassettes.

The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second nucleotide sequence comprising at least one parvoviral (AAV) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.