One variation of a method for producing a target compound includes: during an initial period, genetically modifying a population of insects to produce a target compound; during a growth period succeeding the initial period, cultivating the population of insects; during a treatment period succeeding the growth period, applying a dosage of a stressor to the population of insects, the stressor configured to trigger production of the target compound; in response to a proportion of the target compound within the population of insects exceeding a threshold proportion, harvesting the population of insects; homogenizing the population of insects to form a blend comprising the proportion of the target compound and a second proportion of a set of secondary components; and separating the proportion of the target compound from the second proportion of the set of secondary components for extraction of the proportion of the target compound from the blend.

The application describes methods for synthetic synthesis and cell-free synthesis of DNA vectors, particularly closed-ended DNA vectors (e.g., ceDNA vectors) having linear and continuous structure for delivery and expression of a transgene. The present invention relates to an in vitro process for production of closed-ended DNA vectors, corresponding DNA vector products produced by the methods and uses thereof, and oligonucleotides and kits useful in the process of the invention. DNA vectors produced using the methods described herein are free from unwanted side effects due to contaminants introduced during production in cell lines, for example, bacterial or insect cell lines. Further provided herein are methods and cell lines for reliable gene expression in vitro, ex vivo and in vivo using the ceDNA vectors synthesized using the methods herein.

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 (AAV) 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.

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.

Disclosed herein are recombinant baculoviruses suitable for introducing an exogenous gene into a pest insect, particularly, disease-transmitting mosquitos. The recombinant baculovirus is characterized in having a promotor that is any of a HzNV-1 viral early expressing gene pag1, a ceropin gene b1, a defensin gene a4, or hp70 gene; and an exogenous gene operably linked thereto the promoter. Also disclosed herein is a method of introducing an exogenous gene into a pest insect. The method includes transducing the pest insect with a recombinant baculovirus without suppressing the production of microRNAs (miRNAs) in the pest insect, wherein the recombinant baculovirus comprises a promoter of pag1, cecropin b1, defensin gene a, or hp70.

Nucleic acids encoding Parvoviral Rep proteins with a mutated nuclear localization signal (NLS) are provided. Also provided is a nucleic acid comprising a nucleotide sequence encoding a Parvoviral Rep protein with a mutated zinc finger domain and a nucleic acid comprising a nucleotide sequence encoding a Parvoviral Rep protein comprising an amino acid mutation at position 43, 57, 79, 97, 120, 179, 305, 484, 493 or 571 with reference to SEQ ID NO: 2. Nucleic acid constructs and cells, such as insect cells, comprising the nucleic acids are provided as well as a method for producing a recombinant Parvoviral virion using the nucleic acids.

Provided is a gene expression system, suitable for expression in an insect, comprising an insect muscle actin promoter operably linked to a marker gene, which overcomes or ameliorates one or more of: cost of rearing; amount of handling; errors in identification due to human error or loss of marker by the insect; and health concerns related to the effects of marker powders on workers in mass rearing facilities.

The present inventors have found that by cultivating insect cells, into which the Cas3 gene has been introduced, at relatively low temperatures, it is possible to efficiently express recombinant Cas3 proteins with maintained activity, and by purifying the soluble fractions of these cells, it is possible to collect active forms of the recombinant Cas3 proteins in high purity and high yield.