DNA CONSTRUCTS FOR MANUFACTURING BIO-THERAPUETIC POLYPEPTIDES FOR USE IN ANIMAL VACCINES AND THERAPEUTICS

Abstract

A nucleic acid constructs including a heterologous nucleotide sequence operably linked to a constitutively active promoter; a 5 recombination sequence at a 5 end of the construct and a 3 recombination sequence at a 3 end of the construct, wherein the recombination sequences are configured for homologous recombination into a genome of a microalgal host cell; and optionally a bacterial 5 untranslated region (5UTR) between the 5 recombination sequence and the promoter, and a bacterial 3 untranslated region (3UTR) between the 3 recombination sequence and the heterologous nucleotide sequence.

Claims

1. A nucleic acid construct comprising: a) a heterologous nucleotide sequence operably linked to a constitutively active promoter; and b) a 5 recombination sequence at a 5 end of the construct and a 3 recombination sequence at a 3 end of the construct, wherein the recombination sequences are configured for homologous recombination into a genome of a microalgal host cell.

2. The nucleic acid construct according to claim 1, wherein the heterologous nucleotide sequence comprises a sense sequence joined to an antisense sequence by a linker sequence configured to form a loop structure after transcription.

3. The nucleic acid construct according to claim 1, wherein transcription of the heterologous nucleotide sequence produces a double stranded RNA (dsRNA).

4. The nucleic acid construct according to claim 3, wherein the dsRNA is configured to silence VP28 translation.

5. The nucleic acid construct according to claim 1, wherein the promoter is selected from the group consisting of a T7 promoter, a T5 promoter, and a T4 promoter.

6. The nucleic acid construct according to claim 1, further comprising: a bacterial 5 untranslated region (5UTR) between the 5 recombination sequence and the promoter; and a bacterial 3 untranslated region (3UTR) between the 3 recombination sequence and the heterologous nucleotide sequence.

7. The nucleic construct according to claim 6, wherein the 5UTR is a 5UTR of one or more selected from the group consisting of 16SrRNA, psbA, psaA, rbcL, atpA, atpB, and rps16.

8. The nucleic acid construct according to claim 6 or 7, wherein the 5UTR is complementary to a plurality of ribosomal binding sites.

9. The nucleic acid construct according to claim 6, wherein the heterologous nucleic acid sequence encodes a polypeptide therapeutically active as a vaccine against white spot disease in fish.

10. The nucleic acid construct according to claim 6, wherein the heterologous nucleic acid sequence encodes an antigen from Nervous Necrosis Virus capsid protein (VP).

11. The nucleic acid construct according to claim 6, wherein the heterologous nucleic acid sequence encodes a polypeptide selected from the group consisting of ospA, rOmpB, VP1, VP2, VP4, and VP5.

12. A nucleic acid construct comprising: a) a multicloning site configured for cleavage by a plurality of different restriction endonucleases; b) a constitutively active promoter that directs transcription towards the multicloning site; c) a 5 recombination sequence at a 5 end of the construct and a 3 recombination sequence at a 3 end of the construct, wherein the recombination sequences are configured for homologous recombination into a genome of a microalgal host cell; and d) a bacterial 5 untranslated region (5UTR) between the 5 recombination sequence and the promoter, and a bacterial 3 untranslated region (3UTR) between the 3 recombination sequence and the multicloning site.

13. A vector comprising a nucleic acid sequence comprising the nucleic acid construct according to any one of claims 1-11.

14. A host cell transformed with the nucleic acid construct according to any one of claims 1-12 or the vector according to claim 13.

15. A biomass comprising the host cell according to claim 14 in a dried form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a schematic depicting an exemplary DNA nucleic acid construct joined to a chloroplast transformation vector, and the formation of therapeutic double stranded RNA (dsRNA) structures from transcribing the construct in microalgae.

[0017] FIG. 2 is a schematic depicting a DNA nucleic acid construct incorporated into a chloroplast transformation vector and configured to undergo homologous recombination in microalgae to produce VP28 dsRNA at high copy number for the treatment of white spot in aquatic animals.

[0018] FIG. 3 depicts an exemplary sequence of VP28 sense DNA joined to VP28 antisense DNA by linker DNA (SEQ ID NO: 1), where the underlined portions are the VP28 sense and antisense sequences and the bold sequences are the linker.

[0019] FIG. 4 is a table of viral proteins that can be targeted using the nucleic acid constructs according to the invention.

[0020] FIG. 5 is a schematic depicting an exemplary nucleic acid construct joined to a chloroplast transformation vector, and the resulting high production of therapeutic proteins due to a high copy number of mRNA transcripts from a strong promoter and robust translation by 5UTR enhancers.

[0021] FIG. 6 is a photograph depicting results from northern blotting with corresponding calculated relative transcription rates of chloroplast mRNAs in C. reinhartii cells using the promoters rrn, psbA, rbcL, psaB, atpA, atpB, tufA, rp/16, and pUC.

[0022] FIG. 7 depicts a synthetic construct (SEQ ID NO: 2) formed by fusing the 463bp Chlorella 16S rrn promoter to the T7g10 5UTR.

[0023] FIG. 8 depicts a 367 bp sequence from Chlorella vulgaris, useful as a 3UTR (SEQ ID NO: 29).

[0024] FIG. 9 is a table showing a list of chloroplast promoter sequences which have homology to prokaryotic bacterial promoter elements for use in the invention.

[0025] FIG. 10 depicts an overview of the isolation and processing of VP28 encoding sense and antisense DNA for use as a heterologous nucleotide sequence in the nucleic acid construct.

[0026] FIG. 11 depicts the amplification of restricted sense and antisense VP28 nucleic acid sequences.

[0027] FIG. 12 is an annotated photograph of an agarose gel confirming the presence of the nucleic acid construct with VP28 insert in Colony #6 and a vector map depicting the site of restriction for testing.

[0028] FIG. 13 demonstrates the arrangement of sense and antisense VP28 configured to form dsRNA and flanked by recombination sequences configured for homologous recombination with Chlorella vulgaris.

[0029] FIG. 14 demonstrates a summary of a cloning strategy followed to generate a vector with expression cassette configured for homologous recombination with Chorella vulgaris and expression of green fluorescent protein (GFP).

[0030] FIG. 15 is a vector map with nucleic acid construct with a heterologous nucleotide sequence, which encodes green fluorescent protein, operably linked to a constitutively active promoter in the form of 16S rrn; 5 and 3 recombination sequences configured for homologous recombination into a genome of a microalgal host cell; and a bacterial 5 untranslated region (5UTR) and a bacterial 3 untranslated region (3UTR).

[0031] FIG. 16A is an annotated photograph of an agarose gel after PCR amplification of Chlorella 16S rrn: T7G10 promoter confirming insertion of the 5UTR and promoter in the construct.

[0032] FIG. 16B is an annotated photograph of an agaorse gel after PCR amplification of co-op smGFP confirming insertion in the construct.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0033] The invention provides DNA constructs for the production of recombinant proteins. In particular, the DNA constructs are adapted for integration into microalgae and result in high protein yield, at least in part due to constitutive promoters that allow the microalgae to be grown/manufactured by fermentation technology. Therefore, unlike conventional microalgal systems, the constructs herein do not require sunlight. The fact, that the DNA constructs allow protein expression in the absence of light, the manufacturing process is commercially feasible for large and economic scale, provides batch to batch consistency and is independent of seasonal variation.

[0034] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein as such may vary.

A. Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. In the event that there is a plurality of definitions for terms herein, those in this section prevail. All patents, patent applications, publications and published nucleotide and amino acid sequences (e.g., sequences available in GenBank or other databases) referred to herein are incorporated by reference.

[0036] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods, compounds, compositions described herein.

[0037] The term heterologous as used herein refers to a nucleotide sequence or a polypeptide that is non-naturally occurring in the host cell that is to be transformed or transfected with the nucleic acid construct. A heterologous nucleotide sequence may augment the expression of a protein of interest from an endogenous equivalent gene, i.e. one which normally performs the same or a similar function. A heterologous nucleic acid may comprise a coding sequence of, or be derived from viral origin (e.g. a viral envelope protein).

[0038] The term interfering RNA or RNAi as used herein refers to an RNA molecule that binds a messenger RNA (mRNA) molecule to interfere with or decrease translation of the mRNA into protein. The invention includes the formation of double stranded RNA (dsRNA) to silence or reduce the expression of viral disease.

[0039] The term promoter, as used herein refers to a recognition site on a strand of DNA to which RNA polymerase binds. The promoter is usually a DNA fragment of about 100 to about 200 base pairs. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed enhancers. A constitutively active promoter is an unregulated promoter than remains on.

[0040] The term operably linked as used herein refers to joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.

[0041] The term recombination sequence as used herein refers to a nucleic acid sequence configured to undergo homologous recombination with genomic DNA of a host cell.sub.; preferably a microalgal cell. Recombination sequences are positioned at opposing ends of constructs that are to be incorporated into the genome of the host cell for homologous recombination.

[0042] The term homologous recombination as used herein refers to a reaction between any pair of DNA sequences having a similar sequence of nucleotides (homologous sequences), where the two sequences interact (recombine) to form a new recombinant DNA species. The frequency of homologous recombination increases as the length of the shared nucleotide DNA sequences increases, and is higher with linearized plasmid molecules than with circularized plasmid molecules. Homologous recombination can occur between two DNA sequences that are less than identical, but the recombination frequency declines as the divergence between the two sequences increases. The term configured for homologous recombination into a genome means the 5 and 3 recombination sequences of the construct are sufficiently similar to nucleic acid sequences within the receiving genome that a homologous recombination event can occur.

[0043] The terms 5 and 3 as used herein refer to the relative position along a DNA molecule, where 5 is conventionally upstream and 3 is conventionally downstream in regards to the direction of transcription.

[0044] The term linker as used herein refers to DNA which contains the recognition site for a specific restriction endonuclease. Linkers may be ligated to the ends of DNA fragments prepared by cleavage with some other enzyme. In particular, the linker provides a recognition site for inserting the heterologous nucleic acid, which contains a specific nucleic sequence to be transcribed and potentially translated. When configured to link sense to antisense nucleic acid strands, the linker is configured to avoid complementary binding to each strand so that the linker forms a loop connecting a double stranded molecule characterized by its sense and antisense strand. This recognition site is preferably an endonuclease restriction site, such as, but not limited to EcoR1, HindIII, XhoI, NheI, Sfi-I, and others known in the art to which the invention belongs.

[0045] The term sense sequence as used herein refers to a strand of DNA that has a same sequence as a corresponding mRNA, and the term antisense sequence as used herein refers to an antisense strand of DNA, which acts as template during transcription, and can undergo translation into a protein. The antisense strand is therefore responsible for the RNA that is later translated to protein, while the sense strand possesses a nearly identical makeup to that of the mRNA. The sense and antisense sequences are complementary and can be aligned in opposite directions on a same DNA strand as heterologous nucleotides to cause complementary binding after transcription. Complementary binding can be encourage by way of a linker that links the two sequences.

[0046] The term untranslated region as used herein refers to either of two sections, one on each side of a coding sequence on a strand of mRNA. If it is found on the 5 side, it is called the 5UTR (or leader sequence), or if it is found on the 3 side, it is called the 3UTR (or trailer sequence). The 5 UTR is upstream from the coding sequence. Within the 5 UTR is a sequence that is recognized by the ribosome which allows the ribosome to bind and initiate translation. The 5 UTR in the instant invention acts as an enhancer to increase translation. A bacterial untranslated region does not require the untranslated region be excised from bacterial but instead refers to a nucleic acid sequence that is also found in bacteria or is a nucleic acid sequence having sufficient sequence similarity to a sequence found in bacteria that would result in a same effect in bacteria, such as increasing translation.

[0047] The term therapeutically active as used herein refers to the ability to achieve an improvement in an animal suffering from a disease or reducing a risk of developing a disease. Nonlimiting examples of such improvements include increased survival rate, more rapid recovery, or improvement or elimination of symptoms, and other indicators as appropriate determining measures by those skilled in the art.

[0048] The terms effective amount or therapeutically effective amount, refer to a sufficient amount of recombinant microalgae or therapeutic agent described herein administered which will relieve to some extent one or more of the symptoms of the disease, disorder or condition being treated.

[0049] The term multicloning site as used herein refers to a sequence of DNA having more than two endonuclease restriction sites.

[0050] The term vector as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.

[0051] The term expression cassette as used herein refers to the genetic material of interest which codes for a protein or RNA. The expression cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein in the transformed cell. Preferably, the cassette has 3 and 5 ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end.

[0052] The term amino acid as used herein refers to the molecules composed of terminal amine and carboxylic acid functional groups with a carbon atom between the terminal amine and carboxylic acid functional groups sometimes containing a side chain functional group attached to the carbon atom (e.g. a methoxy functional group, which forms the amino acid serine). Typically, amino acids are classified as natural and non-natural. Examples of natural amino acids include glycine, alanine, valine, leucine, isoleucine, proline, phenylananine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartate, and glutamate, among others. Examples of non-natural amino acids include L-3,4-dihydroxyphenylalanine, 2-aminobutyric acid, dehydralanine, g-carboxyglutamic acid, carnitine, gamma-aminobutyric acid, hydroxyproline, and selenomethionine, among others. In the context of this specification it should be appreciated that the amino acids may be the L- optical isomer or the D -optical isomer.

B. Microalgae as a Host for Therapeutic Agents

[0053] The invention provides nucleic acid constructs for integration into the genome of microalgae for the formation of therapeutic agents. Microalgae have the potential to be an extremely effective oral delivery system for vaccines and therapeutics because they possess several key properties that make them a robust platform. Therapeutic nucleotides expressed within the microalgal biomass are protected within the rigid cell wall of the microalga, allowing the therapeutic or vaccine to remain heat and temperature-stable for up to 1.5 years, and allows the therapeutic molecules to survive the harsh acidic environment of the stomach and be absorbed by aquatic organisms (1). Although the technology is promising, the realization of its potential as a viable platform technology has remained a challenge due to issues with commercial scale-up. Virtually all previous DNA expression vectors for use in microalgae describe the use of photobioreactors or sunlight to grow microalgal therapeutics. Here, we present the first DNA expression vector built specifically for microalgal chloroplast fermentation.

[0054] Among the species of microalgae that can be used as a host for expression of therapeutic agents in the invention include, but are not limited to Chloroficeae, Haematococcus pluvialis, Bacillariophyceae, Dinophyceae, Scenedesmu sp, Chlorella sp., Nannochloropris sp, Diatoms, Phaeodactylum tricornutum, Crypthecodinium sp. and Galdieria sp. Preferably, the micralgae is a species that can be be cultured on a large scale using fermentation. In some embodiments, the species is Chlorella vulgaris, which may be chosen for its high heterotrophic growth rate, and its ability to rapidly assimilate a variety of carbon sources such as glucose, galactose, and acetate(2).

[0055] Microalgae can be transformed with the constructs using conventional techniques known in the art, such as glass bead-assisted transformation, PEG-mediated transformation, micro-injection, particle gun-mediated transformation, electroporation, CRRISPR/CAS9 and shuttle protein-mediated plastid engineering. Furthermore the recombinant microalgae are amenable to high density growth using fermentation.

[0056] The therapeutic agents can be conveniently stored by freeze drying the recombinant microalgae itself. When used to deliver therapeutics to fish, crustaceans and other animals that naturally feed on microalgae, the recombinant microalgae can be supplied for natural feeding. The recombinant microalgae and therapeutic agents provide oral vaccination of fish (grouper, pompano, bass, mullet, seabass, snapper, barramundi, cod, catfish, cuppy, eel, cobia, tilapia, founder, turbot, puffer, striped jack, senegalese sole, sea bream) against diseases such as nervous necrosis viruses (NNV).

[0057] In some embodiments, the microalgae is combined with other carriers or agents that facilitate delivery or with other compounds for improved therapeutic effect. When orally delivered, the microalgae can be formulated with an adjuvant to boost immune response. Among these include poly I:C (recombinant dsRNA), Flagellin fusion proteins, imiquimods, CpG, saponins, squalines, mineral oils and surfactants.

C. Nucleic Acid Constructs

[0058] The invention provides nucleic acid constructs having a heterologous nucleotide sequence operably linked to a constitutively active promoter to form an expression cassette; and flanking recombination sequences configured for homologous recombination into the genome of a microalgal host cell. Upon homologous recombination, the recombinant host cell itself transcribes the heterologous nucleotide to RNA. In some embodiments the RNA is embodied as double stranded RNA (dsRNA) and is provided as a therapeutic agent. In such instances, the dsRNA may act as interfering RNA to interfere with translation of viral proteins. Such an embodiment is particularly useful to combat a variety of diseases that occur in aquatic populations, such as fish farms, prawn farms, and others. In other embodiments, the RNA is translated into a protein and provided as a therapeutic agent, which can be delivered by administering the recombinant microalgae itself or isolating the therapeutic agent and forming a therapeutically acceptable formulation for administration. In these embodiments, the invention is particularly useful in the formation of a vaccine against disease found in aquatic environments, such as fish farms, prawn farms, and others.

[0059] As depicted in FIG. 1, in some embodiments, the invention provides DNA constructs for the improved production of dsRNA for use as a therapeutic agent. In such embodiments, the heterologous nucleotide sequence can be embodied as a sense sequence joined to an antisense sequence by a linker, which upon transcription loops to permit complementary binding of RNA strands to form the double stranded RNA molecule.

[0060] White Spot Syndrome is a virulent disease that infects farmed crustaceans and is responsible for at least 1 billion USD of economic damage to the aquaculture industry each year by some conservative estimates (3). RNA interference has shown to be a promising technology to treat and prevent the pathogenesis of this economically devastating disease (4). One of the major setbacks of using RNA interference for treating white spot syndrome is the lack of an effective and economically viable platform to express and deliver the therapeutic RNAs. The nucleic acid constructs and approach solve this problem by providing a nucleic acid construct that can be integrated into the genome of a microalgal host cell and configured to produce high concentration of therapeutic dsRNA using heterotrophic fermentation.

[0061] In furtherance of the above, FIG. 2 is a schematic depicting a nucleic acid construct inserted into a chloroplast vector for the production of VP28 in the formation of a dsRNA for the treatment of aquatic animals against white spot. In addition, FIG. 3 is a DNA sequence of the VP28 sense and antisense sequences joined by a linker (SEQ ID NO: 1), where the VP28 sequences are underlined and the linker is highlighted by bold typeface. Similarly, other viral proteins can also be targeted using the approaches herein. Among those include the viral proteins provided in FIG. 4, where the heterologous nucleotide sequence is configured to bind the viral protein encoded RNA relying at least in part on sequence complementarity. Configuring heterologous nucleotide sequences to bind viral encoded RNA can be accomplished using conventional molecular biology tools known in the art, such as those that reverse translate polypeptide sequences and reverse transcribe RNA sequences.

[0062] As depicted generally in FIG. 5, in some embodiments the production of a therapeutic protein is desired. In such embodiments, the heterologous nucleotide sequence can be a DNA sequence encoding the therapeutic polypeptide or protein operably connected to a promoter. Determining a DNA sequence that encodes a therapeutic polypeptide or protein can be performed using conventional molecular biology tools known in the art, such as those that reverse translate polypeptide sequences and reverse transcribe RNA sequences.

[0063] In some embodiments the heterologous nucleotide for expression as a polypeptide is a therapeutic agent against Viral Nervous Necrosis. In such embodiments, an immunogenic antigen from the Nervous Necrosis Virus capsid protein (VP) can be encoded by the heterologous nucleotide sequence to provide a vaccine against the disease in several different species of fish, including (but not limited to) warm water species like tilapia and grouper. Other diseases for which recombinant antigens can be produced to illicit an immune response include (but are not limited to) Salmonid Rickettsial Septicemia (antigenic peptides ospA, rOmpB), Infection Pancreatic Necrosis (antigenic peptides for capsid proteins and polymerases, VP1, VP2, VP4, VP5), Viral Nervous Necrosis (antigenic peptides for capsid proteins and polymerases, alpha, and NNV VP), streptococcus agalactiae infection (antigenic peptide GapA).

[0064] Among the antigens that can be targeted for heterologous nucleotide sequence are provided in TABLE 1.

TABLE-US-00001 TABLE 1 Antigens for encoding in the heterologous sequence Bacterial/Viral Disease Antigen Name Uniprot/NCBI Accession Number Nervous Necrosis Viral Capsid Protein (CP) UniProtKB-A0A0F7GYD4 Virus Nervous Necrosis RNA-directed RNA UniProtKB-Q993M1 Virus polymerase Salmonid Rickettsial lipoprotein antigen Genbank (No. CP013821.1) Septicimia (ospA) Salmonid Rickettsial Outer membrane protein B UniProtKB-Q9KKA3 Septicimia (rompB) Infectious Pancreatic RNA Directed RNA UniProtKB-P22173 Necrosis Virus Polymerase (VP1) Infectious Pancreatic Viral Protein 2 (VP2) UniProtKB-Q703G9 Necrosis Virus Infectious Pancreatic Viral Protein 4 (VP4) UniProtKB-M9VYM2 Necrosis Virus Infectious Pancreatic Viral Capsid Protein 5 (VP5) UniProtKB-Q6U2P6 Necrosis Virus Streptococcus glyceraldehyde-phosphate GenBank (No. AFS46447) agalactiae dehydrogenase protein (GapA)

[0065] The promoter drives transcription of the heterologous nucleotide sequence. Preferably, the promoter is chosen based on how well it binds with Plastid Encoded Polymerases (PEPs) and with sigma70-like transcription factors so that the heterologous nucleotide sequence can transcribed at high copy number. These two measurements are strong indicators for how efficiently a gene is transcribed into RNA.

[0066] In contrast to conventional microalgal systems, the nucleic acid construct makes use of a strong, constitutive promoter sequence whose activity is not regulated by light. A plurality of promoters were compared for their efficiency at transcription; the results of which are depicted in FIG. 6. In particular FIG. 6 shows the relative transcription rate of 16S ribosomal rRNA (rrn) is 55 times higher than atpA. Accordingly, in a preferred embodiment, the 16S rrn promoter is provided to drive transcription in the nucleic acid construct. FIG. 7 depicts the 463 bp 16S rrn promoter region (shown in bold) upstream of 16S rrn in Chlorella vulgaris (SEQ ID NO: 2).

[0067] In other embodiments, promoters having lesser efficiency than the 16S rrn promoter may be used; however, in embodiments where protein expression is desired, the invention may include further enhancers within the untranslated regions to increase production. As a nonlimiting example, rbcL was shown to also have a higher efficiency than atpA and thus can also be used. The rbcL gene encodes the RuBisCo large subunit to support the dark reactions of photosynthesis and are generally always expressed. One skilled in the art can also use promoters from core housekeeping genes within the chloroplast.

[0068] TABLE 2 is depicted in FIG. 9, which provides chloroplast promoter sequences having homology to prokaryotic bacterial promoter elements. These sequences are homologous to promoters in other plants, such as microalgae and can therefore be incorporated into the nucleic acid construct. Note the -10 and -35 bacterial promoter consensus sequences which also appear in these chloroplast promoters (TTGACA and TATAAT) (10). SEQ ID NOS: 1-18 and additional chloroplast promoters for use with the invention may be found in Kung, S. D. and Lin, C. M. (1985) Chloroplast promoters from higher plants. Nucleic Acids Res, 13, 7543-7549.

[0069] Alignment with chloroplast genome of the green microalga, Chlamydomonas reinhardtii shows the same conserved -35 and -10 motifs found in higher plant chloroplasts and bacterial promoters. The same homology is observed in microalgae.

[0070] In embodiments where the heterologous nucleotide sequence is to encode a therapeutic protein, the invention preferably includes bacterial untranslated regions to increase translation, thereby increasing protein yield. Most preferably, a bacterial 5UTR is positioned between the 5 recombination sequence and the promoter and the bacterial 3UTR is positioned between the 3 recombination sequence and the heterologous nucleotide sequence.

[0071] Selection of 5UTR is based on mRNA structural stability and ribosome binding strength. In one embodiment of the invention, the bacteriophage T7g10 5UTR/enhancer sequence is used to build a DNA vector to express therapeutic proteins, since it is a highly translationally active 5 UTR regulatory element which has been shown to produce high expression levels in the chloroplasts of higher land plants, and is not downregulated by the presence or absence of light or a particular substrate.

[0072] The 5UTR, when transcribed, has nucleotide elements with homology to conserved prokaryotic ribosomal binding site sequences (typically AGGAGGU a similar variant) which has complementarity to the RNA binding region of the plastid 16S ribosomal RNA sequence.

[0073] Other candidate 5UTR elements include the 5 UTR of psbA, psaA, rbcL, atpA, atpB and rps16. Multiple ribosomal binding sites can also be inserted to increase the rate of translation, increasing the product yield. TABLE 3 provides exemplary 5 UTR sequences for use in the invention.

TABLE-US-00002 TABLE3 5 UTRsequences SEQIDNO: 5UTR Origin Sequence 19 T7g10 T7Bacteriophage GGGAGACCACAACGGTTTTCCCaCTAG AAATAATTTTGTTTAACTTTAAGAAGGA GATATACAT E.coli16S Bacteria ACCTCCTTA rrn 20 rbcL Chloralla gatgaaaaaaagatcaaaaagaaaaaaaagctagag vulgaris aaaatcaaggtttgaaaaaaaatattttttttcatgccttgat Chloroplast tttctttagaaagagaaaaataatgaatttttcaacagtag aacagaatcaaaagaatttcactcttgtgatactctaaaa aatatgctatactctggtgtaaaacaaaaagacaagaa attacactttttttcgggcagagtgcaagatcgtaaaccat aattttttttaga 21 psbA Chlorella aaaaattaaaatgttttttcttttttcctaaaaaggagtttttcc vulgaris caaaattaaaaaaatttaatttgttaggccctactgtaacta Chloroplast aaatctaaatctcagtcaagtaactattaaaataaagatt aaaataaacaaaaaggctactcgatttaaaattaggttttt atattaatttgaaaaactttttttaacaagtaaaaaaagaa gaaaaaatactctaattcttaagattttcaacacataaatt 22 atpA Chlorella ggtaCttctgaactgcctcctctttgttacaaaactcgacgt vulgaris tttgccattcatattgtattaactggctttagaattcgtaaaaa Chloroplast gcacaatttgaaaattattcactgcactatgaattgaagaa gttaatcgttgattaagtttttcgcgaacttgttgtaatgccag ttttactaatttttccgcaagttcattttgagctttttgttgctcaa actttaaggtctcctgttggagagtacctaaacgttttatatc ttcttgtgtttggcgaatgaattg 23 rpoC1 Chloralla gaaaaaaaaacggaatcagaaaagaaagaaatgac vulgaris aaaaaaatgatttttttcattccattttttttttagaaaaaaaa Chloroplast acgaaaattaaggcatttatttttttaaggatcaaaaagatt tttttttcaagcctatctttttttttctaaaaaaaaagaagggt ggaacccttcattttctgattccattttttttttttaagaaagaa aatcttgattttagaacttttttagctcttttttaaattgggctttt gtttgatattcttttgtacaaattaaagaaaacactaaaacc tttccaccctaagt 24 psaA Chlorella gtttcatttaaacgaaaattgtactgagtgttttaaccaaaa vulgaris tttaagcacctttgcagatttcgtcatctttcttttttttgaaaaa Chloroplast aaaagaaataaaaagaaaaaaagctgttttaaactgaa tagtaactaaccctttatgcaccagagttctatgggatcct ataacgaaactctcaagttactcttttgtgagtacaatcaa acgcaagactcatatttctgtctagtgattttgtaaatttttgg aactaccaaaaattagaacttttggtttcaaatttcaactttg tcattattttttttgattttcttaaccgctattttttttaaaaatactg tggtaagattttaaaaaagataatttcggtaattcaaataa ggagaatttttcgcc 25 T7g1.3 Bacteriophage tacgactcagtatagggacaatgcttaaggtcgctctcta ggagtggccttagtcatttaaccaataggagataaacatt 26 psbH Chlorella aatttcaagggtagtagaattttttttactttctgtaacgaga vulgaris aaactaaaagaaagttttataaattttaaatcattttagttttc Chloroplast gattttaacgtcaattttttcagactttcttccaattttattatata gtaaattttaaattggaaaaaggttttagttacttctttcgaa aacgcaactaaaagtataaaatgttttttgaaaacaaaat ttttagtttctttaaagttttta

[0074] The selection of the 3UTR is based on structural stability. Generally, the choice of 3UTR has little effect on the expression of the gene. In most embodiments of the invention, the 3UTR of rbcL is chosen. In other embodiments, the 3UTR of 16S rrn of Chlorella vulgaris (SEQ ID NO: 27 and shown in FIG. 8 may be use as the 3UTR. In yet other embodiments, one can use the 3UTRs of atpA, psA or others. TABLE 4 provides exemplary 3 UTR elements.

TABLE-US-00003 TABLE4 3 UTRsequencesfromC.vugaris SEQIDNO: 3UTR Sequence 28 rbcL ttttcaactaaactgttccagttctaaagaaaattaaggaaaat ccttaattttctttaactttttttctttttttttcttctttttgatcttttttttttta taaaaaaaagaaaaaggaaaaaaaagatcaaaaaggtc tacttaaaaacataaatgccttgcattt 29 16srrn ccttttaaaggataaaaaaaccttaagaaaaaacaaagttttt cttaagtactccaatccttctttctttttttattcccattaaattgaat aaaaattaaacgttttcttactttaatgagaatataaattaagat gatttccgttttttgaaatccagtttttggttttttttttccttctttttttttt ataaaaaaaaagatcaaaatgaatcaaaaactaatagaa aaaaacggaatttgaagaattgattcattttttacatgtgcatga gtcactagcttgcttttttagtgcactaacctcttctcca 30 atpA aaaagtatgttttattagtaaagtatcagatttatacagagctta ataaaaacagggcccaataaatgaatgggaaaaattttaaa ttatgttttttttacttttgatctttttttttagttcttttttttctttttttttttttga aaaaaaaaagaaaaaaaagaactaaaaaaaaagatca aaagtaaaaaaaacataaccaaaaaataccagcaaagc atgtatttttttaagggttgagatacttagtt 31 psaA gaaaaccaggcaaaaaatttcgtttctcttactttaaagaattg gaaaggtttctgattccacaaaaaaaatgcgaagcattttttta taattcctttttttgctcttcctaaaaaaaaaatgcaaagcattttt ttttaggataatttctgattctttttgatcttttttttttagttctttttgatgt ttttttttttataaaaaaaaaacaaatagaaaaaaaagaaaa aaaagaagcaaaaaaagatcaaaaaggaaatagtaggc aggtttccaacttaaaaaattaaaagcaaagctt

[0075] The choice of 5 and 3 homologous recombination sites used to integrate the construct into a chloroplast can affect expression. Different regions of the chloroplast genome can have different levels of expression. The most transcriptionally active intergenic spacer regions are used to create the construct. In one embodiment of the invention, homologous recombination sites from the chloroplast of a species of heterotrophic microalga, Chlorella vulgaris, are chosen in order to integrate the DNA Expression Cassette into a transcriptionally active intergenic spacer region. This region is located between rbcL and trnS within the chloroplast genome. Other transcriptionally active spacer regions that are generally used for high expression and thus are a target for homologous recombination include include trnI/trnA, rps14/trnG, trnP/rps12.

D. Therapeutic Treatment

[0076] In some embodiments the construct is configured to produce a therapeutic agent embodied as a dsRNA configured to interfere with viral DNA. In other embodiments, the therapeutic agent is used as a vaccine for animals, and in particular aquatic animals such as fish and crustaceans. Microalgae provides not only an efficient host for therapeutic production but also provides an effective delivery vehicle, thereby reducing the complexity of the therapeutic formulations themselves.

[0077] Whether the therapeutic agent is delivered by freeze dried tablet or powder or whether combined with subsequent agents, the therapeutic agent is provided to the animal population in a therapeutically effective amount or a prophylactically effective amount. It is understood that an effect amount can vary from species to species due to variation in metabolism of the microalgae or the therapeutic agent, genetics, composition, age, weight, general condition of the species, the condition being treated, the severity of the condition being treated, and the judgment of those having skill in the treatment of such species.

[0078] To demonstrate the bioavailability of orally delivered vaccine antigens expressed in microalgal chloroplasts to fish, codon-optimized green fluorescent protein (GFP, 717bp) was cloned into a chloroplast expression vector as described in Example II.

EXAMPLES

Example I

Construction of a Vector with Expression Cassette for Integrating White Spot Syndrome Virus VP28 dsRNA Coding Sequence Into the Genome of Chlorella vulgaris

[0079] Serving as a DNA vector backbone, pASapI was provided courtesy of MicrosynbiotiX's scientific advisor, Dr. Saul Purton (5). As depicted pictorially in FIG. 10, VP28 dsRNA coding sequence with linker (SEQ ID NO: 1) was synthesized then cut with the restriction enzymes XbaI, EcoRI, and BlpI, to produce two digested VP28 sense and antisense fragments with 5 and 3 overhangs. As shown in FIG. 11, the DNA fragments were amplified using a forward primer SEQ ID NO: 32 and reverse primer SEQ ID NO. 33 to create the appropriate overhangs for the VP28 sense fragment, while another set of primers, Forward Primer SEQ ID NO: 34 and Reverse Primer SEQ ID NO: 35 were used to create the appropriate overhangs for the VP28 antisense fragment.

TABLE-US-00004 TABLE5 PrimersforVP28fragmentamplification SEQIDNO: Primer Sequence 32 VP28sense TAAAATTCTCGAGATGGATCTTTCTTTCACT forward CTTTCGGTC 33 VP28sense CCGAGTAAaagggcgaattcgccctt reverse 34 VP28antisense CCGAGTAAaagggcgaattcgccctt forward 35 VP28antisense gagtacaagcttATGGATCTTTCTTTCACTCTTT reverse CGG

[0080] The sense and antisense fragments, with complementary overhangs within the linker region, were ligated together using T4 DNA Ligase to produce the palindromic dsRNA gene sequence with restriction sites XhoI and HindIII. The recipient vector (pASapI) was then cut with restriction enzymes XhoI and HindIII to create complementary overhangs. The fragment was then ligated into the pASapI vector.

[0081] Cloning of chlorella 16S rrn promoter into DNA Expression Cassette. The chlorella 16S rrn promoter (SEQ ID NO: 36) was synthesized using Thermofisher Gene Art Service and cut using restriction enzymes MluI and XhoI. The same restriction enzymes were also employed to cleave the existing atpA promoter and its 5UTR from the DNA backbone (pASapI). T4 DNA ligase was then used to assemble the restricted chlorella 16S rrn into the DNA backbone.

TABLE-US-00005 TABLE6 SyntheticChorella16Srrnpromoterregion SEQIDNO: Description Sequence 36 SyntheticChlorella16S taaagaaaagtgagctattaacgcgtATCAAAGTTAAATT rrnpromoterpriorto CGTAAATTTTAACTTTGATTCTTCTGGAAGTGC restriction AACTACTTTCCGTTTAGCTGGTTGAAATCTTTA GTATAAATTTTTTTTTAAGCAAAAAATTTAATTT AACTATGATAAATTTAAAAATTAAAGCAAGGAA AAAAAGAAGAGAGAGGAAGACTGGTTTGACTT TTTTTTTATTAAAGATACATTACTAAGTGTGAAA ACAaagcttgtactcaagctcgtaacg

[0082] Replacement of pASapI flanking regions for homologous recombination. Lastly, the 5 and 3 recombination sequences (SEQ ID NOS: 37, 38) were amplified from the chlorella genome and cloned into the Expression vector. The 5 Recombination site (SEQ ID NO: 37) was amplified from chlorella genomic DNA using Forward Primer containing Pcl1 cut site SEQ ID NO: 39 and Reverse Primer containing the Mlu1 cut site SEQ ID NO: 40. The resulting PCR product and the pASapI backbone were then digested with Mlu1 and pcl1, and the overhangs of the insert and the template were joined together using T4 DNA ligase. A similar procedure was employed for the 3 Recombination site (SEQ ID NO: 38), using Forward Primer containing Afe1 restriction site (SEQ ID NO: 41) and a Reverse Primer with restriction site KasI (SEQ ID NO: 42) to amplify the desired region from chlorella vulgaris genomic DNA. The fragment and the insert (pASapI) were both digested with Afe1 and KasI, and the complementary overhangs were joined together using T4 DNA ligase.

TABLE-US-00006 TABLE7 Insertionofrecombinationsequences SEQIDNO: Description Sequence 37 Chlorellavulgaris5 ATAAAAAAAAGAAAAAGGAAAAAAAAGATCAAAA RecombinationRegion AGGTCTACTTAAAAACATAAATGCCTTGCATTTA TGTTTTTAAGGATAAAAAATATTTTTGGCCGTCT TTCTTTTCTTAAGCTTTTTTTTCAAAAGCTTAAGA AAAGAAAAGATTTTCCTTTATTTTTTTTAGAACCG GAACAAACTTTTGAAAAAAAATTTACTTTTTTTTC ATCAGTCCTCCATTGAAAATGGAGAGTCCCCTT CTTTTTTTTTATAAAAAAAAGAATGAAAAGAAATG CTTTGTATTTCTTTTCTTTAGGAAAAAATAAAATT TTTTCATTTTAACTGATACTAAAATTCGTTTCTTT TTTTTGTGTAATAAAATAAAAAAGAAATAAATAAC TGAACCTAGATATGGCAAAAAAAAGCATGATTG AGCGTGATAGAAAACGCGCTCGTTTAATTACTA AATATGCAGCAAAACGAAAAAATCTCCTCGTAG AAATTAAAACAGCAACTTCTTTAGAAGACAAATT CAATTTACATAGAAAATTGCAACAACTACCAAGA AATAGTGCACCAGTTCGATCTCATAATCGTTGTA CAATTACCGGTCGGCCCAGAGGATATTTTAGAG ACTTCGGCTTATCCCGCCATGTTTTACGCGAAT ATGCTTTACAAGGTTTTTTACCTGGTGTGGTAAA AGCTAGTTGGTAAAAAAACTGTAGTTGACCTATT GACTTTTGATTTCCGTTCTTGTCTTTTCTTTTTGA GCTTTTTTTTATAAAAAAAAAAGAAAAAAAAGCT CAAAAAGAAAAACTACACATTATTAAAGCGTCGC GATCTGGGTATCAGAAACAAAAGCGACATGGTG ATTACATAATTTTAAAGGAAAGCTACATGGTGAT TACATGATTTTAAAGGAAAATTACATAGTGATTA CATAACGTTTTTAGCGAATAAACCTTGCCAATAC AGCATTTTTTTCTTTTTTTTTTTTTTTAATGTGATA TTTAAGTCGTTTAATTTTTCAACACGTTTTTTTTA ACAACTTTCGAAATAACAACTTTAACCAACTTGT TTTTAGCCTTACTTAGTTTCAATCAAGAAGTTGG TTTTGAAA 38 Chlorellavulgaris3 TCCTTGTTACGGTGGATTTTTAGCTATTGCGCTT RecombinationRegion TTTCCTTTTTTTTAGTATAATTTTTATTACCTTTTA ACAAGCCTGCTTAGCTCAGTTGGTTAGAGCATC CGTCTCATACGCGGAATGTCACTAGTTCGAATC TAGTAGCAGGCACCATAATTTCGCGGGTATAGC TCAGTGGTAGAGCGGCACCTTGCCAAGGTGCA TGTCGCGCGTTCGACTCGCGTTACCCGCTTATT TTATTTTTTCTATTTAAGTTTTTTCATAAAATCTAA AATTAAAAAGAGTCTATTTAATCCAGTTTTTTGGT TCGAATAGTTTTTCGTTTCTTTTTTTCAAAAAAAA AAAAGAATGAAAAACTCAAAAAACAAAAACGGG TTCCACCCTTAAAAAAAATGCTGTGCATTTTTTT AAGTAAACCCAATGCTTTTTTTTTATAAAAAAAAA GAATGAAAAGAAATTCTTTTTTTTTCATGCCTTTA TTTTCTTTAGAACCGAAAATCAAAGAAAATTTTCT TTTATAAATTAAATCGTTTCTATCTTTTTTTTTCTA AAAAAAAAAGAAGGAAAAAAAATACTTTTTTCAA AGTTATTACGGAATATGTTAAATCGAGGGTGAC GGGACTCGAACCCGCAACTTCCACCGTGACAG GGTGGTGCTCTGACCAATTGAACTACACCCCCT CGATATGGATTTATCATATCACACATCTTTTTTTT AATGTAGAGCTAGCTGTCAGTTCCAGTACAAATT GTCTATTTTATTTATTTTCAAAAGGAGATAAATCT GTCTTTTAATAAGTTTATAGCTGAATAAAAAATTT TTCTCTTACGAAAATAAATGCAAAGCACTTATTT CCTGATTTCGTTTTTTAAATTGAATAGTTTTTTTT CTTTTTTTCAAAAAAAGAAAGAAAAACTCCAGGT TAAAAAACGGCTTTGAAGCGACTTCCGTTTTTTT ATTCAATGAGTTTTTCTTTAGAAAAACTTCTGATT CTTAAAAAAAAAGGTTTGAAAAAAATGATTTTTTT CAAACTTTTCTTTTCTTTAGAAGTCGAAGTCGTA GAAAATTCTGAAGAATTTAAATTTTAAATTCTTTA GCTTAAAAAAAAAATTGTCATTCCT 39 5 flankingregion ACATGTATAAAAAAAAGAAAAAGGAAAAAAAAGA forwardprimer TCAAAAAGGTCTACTTAA 40 5 flankingregion ACGCGTTTTCAAAACCAACTTCTTGATTGAAACT reverseprimer AAGT 41 3 flankingregion AGCGCTTCCTTGTTACGGTGGATTTTTAGCTATT forwardprimer GC 42 3 flankingregion GGCGCCAGGAATGACAATTTTTTTTTTAAGCTAA reverseprimer AG

[0083] E. coli (Dh5a) was transformed with the vector/construct. Individual colonies were manually picked and tested for the presence of a correct VP28 expression cassette by digestion with XhoI+EcoRI+HindIII. FIG. 12 depicts an annotated photograph of the results from colony #6, which displayed bands at the correct sizes (627bp and 529bp).

[0084] A shown in FIG. 13, colony #6 was then sequenced confirming the presence of the nucleic acid construct having a heterologous nucleotide sequence embodied as a VP28 sense sequence joined to a VP28 antisense sequence by a linker sequence, and which is operably linked to a constitutively active promoter embodied as a 16S rrn promoter; and a 5 recombination sequence at a 5 end of the construct and a 3 recombination sequence at a 3 end of the construct, that are configured for homologous recombination into a genome of Chlorella vulgaris.

Example II

Construction of a Vector with Expression Cassette for Integrating Codon-Optimized GFP Into Chlorella vulgaris Chloroplasts

[0085] The pAsapI vector was once again used as the backbone for the cloning experiment. The sequence for 16S rrn with T7g10 5UTR (SEQ ID NO: 43) was synthesized using Thermo Fisher Gene Art service. mGFP was also codon optimized based on the codon usage frequencies from rbcL and psbA of Chlorella vulgaris, which are two most highly expressed chloroplast genes in Chlorella vulgaris, Chlorella variabilis and Chlorella mirabilis, and synthesized using Thermo Fisher's Gene Art Service, and was built with NdeI and HindIII cut sites in the 5 and 3 regions, respectively (SEQ ID NO: 44). An overview of the cloning strategy is depicted in FIG. 14 and the final vector is shown in FIG. 15. The promoter and 5UTR regions were amplified using Forward Primer with a built in cut site for MluI (SEQ ID NO: 45), and Reverse Primer with a built in cut site for HindIII (SEQ ID NO: 46).

TABLE-US-00007 TABLE8 mGFP&5UTR SEQIDNO: Description Sequence 43 Chlorella16Srrn taaagaaaagtgagctattaacgcgtATCAAAGTTAAATTCGTAA (129bp)+ T7G10 ATTTTAACTTTGATTCTTCTGGAAGTGCAACTACTTTC (Proteinexpression) CGTTTAGCTGGTTGAAATCTTTAGTATAAATTTTTTTT TAAGCAAAAAATTTAATTTAACTATGATAAATTTGGGA GACCACAACGGTTTTCCCaCTAGAAATAATTTTGTTTA ACTTTAAGAAGGAGATATACATATGccctaagcttgtactcaa gctcgtaacg 44 Codonoptimizedwith TAACTTTAAGAAGGAGATATACATATGTCTAAAGGAG restrictionsites AAGAGCTTTTTACTGGTGTTGTACCAATCTTAGTTGA ATTAGATGGTGATGTTAATGGGCACAAATTCTCTGTA AGTGGTGAAGGTGAAGGTGATGCAACATACGGTAAA TTAACTCTTAAATTTATTTGTACTACTGGTAAATTACC TGTTCCATGGCCAACATTAGTAACTACTTTCTCTTAT GGTGTTCAATGTTTTTCACGTTACCCAGATCATATGA AACGTCACGACTTCTTCAAATCTGCTATGCCTGAAGG TTACGTTCAAGAACGTACTATTTCTTTCAAAGACGAT GGGAACTACAAAACTCGTGCTGAAGTTAAATTTGAAG GTGACACTTTAGTAAACCGTATTGAGTTAAAAGGAAT CGACTTCAAAGAGGATGGTAATATCCTTGGCCACAA ATTAGAATATAACTACAACTCACACAACGTATACATC ACTGCAGACAAACAAAAAAATGGTATCAAAGCTAACT TCAAAATTCGTCACAACATTGAAGATGGTAGCGTTCA ACTAGCAGATCATTACCAACAAAACACTCCAATTGGC GATGGCCCTGTACTTTTACCAGACAACCATTACTTAT CAACTCAATCTGCTTTATCTAAAGATCCTAACGAAAA AAGAGATCACATGGTATTACTTGAATTTGTAACAGCT GCTGGGATTACACATGGCATGGATGAACTATACAAAT AAaagcttgtactcaagctcgtaacgaa 45 5UTRforwardprimer taaagaaaagtgagctattaacgcgtggcaggcaacaaatttatttatt gtcccg 46 5 UTRreverseprimer cgttacgagcttgagtacaagcttCATATGTATATCTCCTTC TTAAAGTTAAACAAAATTATTTCTAGtGGGAAAAC CGTTGTGGTCTCCCac

[0086] The 16s rrn T7g10 promoter/UTR region, and the pASapI backbone were both digested with MluI and HindIII to remove the atpA promoter in pASapI and to facilitate the joining of the 5 and 3 overhangs of the insert and the construct. The insert was ligated into the pAsapI backbone using T4 DNA ligase. The resultant construct, as well as the mGFP insert, were digested with NdeI and HindIII to facilitate restriction cloning into the DNA backbone. The fragment and the insert were then ligated together using T4 DNA ligase.

[0087] Lastly, the 5 and 3 recombination sequences were amplified from the chlorella genome and cloned into the Expression vector. The 5 Recombination site was amplified from chlorella genomic DNA using Forward Primer containing Pcl1 cut site (SEQ ID NO: 39) and Reverse Primer containing the Mlu1 cut site (SEQ ID NO: 40). Results are shown in FIG. 16A.

[0088] The resulting PCR product and the pASapI backbone were then digested with Mlu1 and pcl1, and the overhangs of the insert and the template were joined together using T4 DNA ligase. A similar procedure was employed for the 3 Recombination site, using Forward Primer containing Afe1 restriction site (SEQ ID NO: 41) and a Reverse Primer with restriction site KasI (SEQ ID NO: 42) to amplify the desired region from chlorella vulgaris genomic DNA. The fragment and the insert (pASapI) were both digested with Afe1 and KasI, and the complementary overhangs were joined together using T4 DNA ligase.

[0089] E. coli (Dh5a) was transformed with the vector/construct. Individual colonies were manually picked and tested for the presence of a correct GFP expression cassette by digestion. Results are shown in FIG. 16B.

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