Patent Grant
11332754
This invention relates to improved transgene expression in chloroplasts through codon optimization using genomic, proteomic and ribosome profiling methods. Transgenes so improved and methods of use thereof are also provided.
Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
A major limitation in clinical translation of human therapeutic proteins in chloroplasts is their low level expression. Prokaryotic or shorter human genes are highly expressed (up to 70% of total leaf protein) in chloroplasts. For example, native prokaryotic genes from Bacillus anthracis and Bacillus thuringiensis were expressed up to 29.4% and 45.3%, respectively, of the total soluble protein (TSP) in chloroplasts (De Cosa et al., 2001; Ruhlman et al., 2010). Small human genes including insulin like growth factor-1 (˜7.8 kDa, Daniell et al., 2009), proinsulin (˜12 kDa, Ruhlman et al., 2010), and interferon-α2b (˜21.5 kDa, Arlen et al., 2007) were expressed in chloroplasts at very high levels. However, expression of larger human proteins presents a major challenge.
Likewise, expression of viral vaccine antigens is quite unpredictable with high, moderate or extremely low expression levels. For example, due to their susceptibility to proteolytic degradation at the N-terminal region, VP6 antigen of rotavirus accumulated at very low levels in tobacco chloroplasts (Birch-Machin et al., 2004; Inka Borchers et al., 2012). The expression level of human papillomavirus-16 L1 antigen varied from 0.1% to 1.5% and accumulated up to 2% when fused with LTB but extremely low when fused with glutathione-S-transferase (GST) in tobacco chloroplasts (Lenzi et al., 2008; Waheed et al., 2011a; Waheed et al., 2011b; Hassan et al., 2014). Further, the instability of chloroplast-derived antigens against human immunodeficiency virus type 1 (HIV-1) has been reported in tobacco. Chloroplast-derived p24 protein only accumulated ˜2.5% in youngest leaves and was not detectable in mature leaves when native p24 cDNA was expressed (McCabe et al., 2008). It is well known that high doses of vaccine antigens stimulate high level immunity and confer greater protection against pathogens and therefore higher level expression in chloroplasts is a major requirement (Chan and Daniell, 2015).
Such challenges have been addressed by the use of optimal regulatory sequences (promoters, 5′ and 3′UTRs), especially species specific endogenous elements (Ruhlman et al., 2010). Cursory attempts have been made to simply increase AT content by modifying third position of each codon of human genes (Daniell et al., 2009). In vitro assay of inserted gene for translation efficiencies of several synonymous codons are not always correlated with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007) but have been used in the past codon optimization studies (Ye et al., 2001; Lenzi et al., 2008; Jabeen at al., 2010) because there are no such in vivo studies. Therefore, no systematic study has been done to utilize extensive knowledge gathered by sequencing several hundred chloroplast genomes to understand codon usage and frequency of highly expressed chloroplast genes. Another major challenge is the lack of reliable methods to quantify insoluble proteins because the only reliable method (ELISA), can't be used due to aggregation or formation of multimeric structures. Targeted Proteomic Quantification by Mass Spectrometry by parallel reaction monitoring (PRM) has become a powerful tool for relative and absolute protein quantitation based on its specificity and sensitivity (Domon and Aebersold, 2010; Gallien et al., 2012). In addition, PRM offers high specificity and multiplexing characteristics which allow specific monitoring of multiple fragment ions of peptides, based on nanoLC retention times and precursor ion m/z (Gallien et al., 2012) but this concept has never been tested for plant protein drugs.
Drawbacks associated with expression of live attenuated and killed viruses include the potential to revert to virulence, low levels of immunogenicity, antigenic variability between species, and possible transfer of genetic materials to wild-type strains (Burns et al., 2014). An outbreak of type 2 vaccine-derived polio (VDVP2) in Nigeria, first detected in 2006, became endemic in Africa and persists today (Famulare et al., 2015). This large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred since 2005 in northern Nigeria; phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 through 2011 resolved the outbreak into 23 independent VDPV2 emergences, at least 7 of which established circulating lineage groups (Burns et al., 2013). Non-polio enteroviruses (NPEVs) associated with acute flaccid paralysis (AFP) cases have been reported frequently through Polio Surveillance Programs (PSPs) worldwide (Laxmivandana et al., 2013). Although wild polio cases have been eradicated in many countries due to intensive oral polio vaccination programs, more non-polio AFP cases are being reported worldwide. Currently recognized EV species have been divided into poliovirus (PV) containing the three PV serotypes and human enterovirus (HEV) A, B, C and D (Dhole et al., 2009). Based on phylogenetic analysis of their genomes, PV and serotypes of the HEV-C species are closely related (Brown et al., 2003). Further, the high frequency of circulation of HEV species C has led to reports of vaccine-derived poliovirus (VDPV) outbreaks (Rakoto-Andrianarivelo et al., 2005). Between 2005 and 2011, 23 lineages of circulating vaccine-derived polioviruses (cVDPVs) with origins in the nonstructural region (NSR) of non-polio enterovirus C (NPEV-C) origin were detected in Nigeria. Thus, recombination between Sabin oral poliovirus vaccine (OPV) and indigenous NPEV-Cs led to some of the recombinant cVDPV lineages isolated during the outbreak in Nigeria (Adeniji et al., 2015). The cVDPVs are largely generated by homologous recombination between OPV and HEV-C and caused numerous outbreaks of poliomyelitis globally, becoming a serious health threat (Jiang et al., 2007). Due to recombination of OPV with HEV-C, highly virulent cVDPVs have the risk to replace wild-type PVs in regions with low vaccine coverage. In an effort for global PV eradication, worldwide cessation of OPV vaccination has been proposed to minimize the number of vaccine-derived poliovirus strains that could lead to new outbreaks (Kouiayskaia et al., 2015; Parker et al., 2015).
Plant-derived subunit vaccines are heat-stable and are free from contamination with animal pathogens. They can also be engineered to contain multiple antigens and transmucosal carrires, to protect against multiple infectious diseases (Chan et al., 2015). Recent report of intact plant cells expressing green fluorescent protein (GFP) between villi of the ileum after oral delivery provided direct evidence for protection of protein drugs in the digestive system from acids and enzymes in the stomach; GFP fused with the transmucosal carrier CTB released into the gut lumen from plant cells was absorbed by epithelial cells via GM1 receptor mediated delivery (Xiao et al., 2015). Such mechanistic and conceptual advances could revolutionize vaccine delivery by eliminating the cost of complex production systems, such as fermentation, purification, cold storage and transportation (Jin et al., 2015 and Kwon et al., 2013. Although potato-derived HBsAg expressed via the nuclear genome was tested in pre-clinical and in human clinical trials a decade ago, (Kong et al., 2001; Thanavala et al., 2005) progress in advancing to later stages is slow. Two major challenges are the low levels of expression of antigens via the nuclear genome and the potential to induce tolerance without injectable priming of antigens with adjuvants (Chan et al., 2015; Rybicki et al, 2014).
In accordance with the present invention, a method for increasing translation of a transgene encoding a protein of interest in a chloroplast is provided. An exemplary method comprises analyzing the native sequence of a nucleic acid encoding said protein of interest and replacing codons in said sequence with those preferentially used in psbA genes in chloroplasts across over 100 plant species and optionally performing ribosome profiling and removing any codons that cause stalling of ribosomes during translation. A synthetic, codon optimized sequence is then produced and cloned into a chloroplast transformation vector, said synthetic sequence being operably linked to 5′ and 3′ regulatory elements for suitable for expression in said chloroplast. Target plants are then transformed with this vector under conditions whereby said therapeutic protein is expressed, wherein replacing said codons causes at least a two fold, three fold, four fold, five fold, 20 fold or 40 fold increase in protein expression relative to expression levels observed using the native sequence. The method can optionally entail isolating said protein of interest. In a preferred embodiment, the method further comprises harvesting and lyophilizing leaves from said plant, wherein the lyophilized leaves comprising the protein of interest.
In particularly preferred embodiments, synthetic VP1 protein is produced that can be used to advantage in vaccines for the treatment of polio. Accordingly, a method of producing systemic and mucosal immunity in a subject who has been previously immunized against polio virus comprising orally administering the lyophilized plant cells described above to said subject in the presence of an adjuvant, said administration causing production of anti-VP1-IgG1 and anti-VP-1-IgA titers in said subject, thereby boosting immunity to said polio virus is provided.
In another embodiment, Factor VIII heavy and light chains have been codon optimized. Factor VIII so produced can be used to advantage in methods for the treatment of coagulation disorders. Thus, the invention also provides for methods for the treatment of coagulation disorders using coagulation factors optimized for efficient expression as disclosed herein. While FVIII is exemplified herein, other coagulation factors, such as FIX, FX, and FVII can readily be optimized using the guidance provided herein.
The methods of the invention can also be used to advantage to produce synthetic insulin growth factor (IGF-1). Methods of treatment of IGF-1 deficiencies using the synthetic IGF-1 described herein are also within the scope of the invention.
In yet another embodiment, a synthetic mutanase enzyme is provided. Methods for treating dental caries using synthetic mutanase enzymes are also disclosed.
In another aspect of the invention, a method of producing systemic and mucosal immunity in a subject who has been previously immunized against polio virus comprising orally administering the lyophilized plant cells described above to said subject in the presence of an adjuvant, said administration causing production of anti-VP1-IgG1 and anti-VP-1-IgA titers in said subject, thereby boosting immunity to said polio virus.
Also within the scope of the invention are plastid transformation vectors encoding the synthetic proteins described herein. Plants comprising such vectors also form an aspect of the invention. In a preferred embodiment, the plant is edible.
In the present invention, heterologous gene expression utilizing chloroplast genome sequences, ribosome profiling and targeted proteomic quantification by mass spectrometry or parallel reaction monitoring (PRM) was employed to develop methods for increasing translation of heterologous proteins of interest in chloroplasts. Codon optimization based on psbA genes from 133 plant species increased translational efficiencies of heavy chain of the human clotting factor VIII (FVIII) and polio viral capsid protein 1 (VP1), when compared with corresponding native genes, driven by identical psbA regulatory sequences. PRM analysis using peptides from N or C terminus showed 5-7 or 22-28 fold increase in FVIII or VP1 codon optimized genes. Western blot analysis of the same batch of materials showed either lower or higher quantitation, underscoring some limitations. PRM is validated here for the first time for quantitation of biopharmaceuticals in plant cells, especially useful for insoluble or multimeric proteins. Despite prokaryotic origin, codon usage is different between E. coli and chloroplasts. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance or stability. Ribosome foot prints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing rate limiting steps. A major ribosome pause at CTC leucine codons in the native gene was eliminated upon codon optimization. Ribosome stalls were observed at clusters of serine codons in the codon-optimized VP1 gene. Synthetic sequences which eliminate CTC leucine clusters further optimizes such sequences.
The WHO's Strategic Advisory Group of Experts recommended complete withdrawal of OPV2 in 2016 globally, replacing with at least one dose of IPV. However, high cost, limited supply of IPV, persistent cVDPV transmission and need for subsequent boosting remain unresolved. The strategy of using a low cost cold-chain free plant-made viral protein 1 (VP1) subunit vaccine as an oral booster after single IPV priming is a novel solution to address this critical need. Oral boosting of VP1 bioencapsulated in plant cells resulted in high VP1-IgG1, IgA and neutralizing antibody titers (˜3.17-10.17 log 2 titer) against all three poliovirus Sabin serotypes. Ability to store lyophilized plant cells expressing VP1 at ambient temperature indefinitely without loss of efficacy eliminates cold chain currently required for all vaccines. These findings provide evidence for plant-made booster vaccine to replace OPV or boost immunity among the elderly population with waning immunity for immunizations received early in life.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, “at least one” means that more than one can be present. Also, the use of “comprise”, “contain”, and “include”, or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting and means “including the following elements but not excluding others.”
The term “consists essentially of,” or “consisting essentially of,” as used herein, excludes other elements from having any essential significance to the combination. Use of “or” means “and/or” unless stated otherwise. The term “and/or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or “X and Y”.
As used herein, the terms “administering” or “administration” of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. The administering or administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administering or administration includes self-administration and the administration by another.
As used herein, the terms “disease,” “disorder,” or “complication” refers to any deviation from a normal state in a subject.
As used herein, by the term “effective amount” “amount effective,” or the like, it is meant an amount effective at dosages and for periods of time necessary to achieve the desired result.
As used herein, the term “inhibiting” or “treating” means causing the clinical symptoms of the disease state not to worsen or develop, e.g., inhibiting the onset of disease, in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.
As used herein, the term “CTB” refers cholera toxin B subunit. Cholera toxin is a protein complex comprising one A subunit and five B subunits. The B subunit is nontoxic and important to the protein complex as it allows the protein to bind to cellular surfaces via the pentasaccharide chain of ganglioside.
A “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control. A replicon may be either RNA or DNA and may be single or double stranded.
A “vector” is any vehicle to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
An “expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
The term “promoter region” refers to the 5′ regulatory regions of a gene (e.g., 5′UTR sequences (e.g., psbA sequences, promoters (e.g., universal Prnn promoters or psbA promoters endogenous to the plants to be transformed and optional enhancer elements.
The term “oligonucleotide,” as used herein refers to sequences, primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
The phrase “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
As used herein, the terms “reporter,” “reporter system”, “reporter gene,” or “reporter gene product” shall mean an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radio immunoassay, or by calorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product. The required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.
The terms “transform”, “transfect”, “transduce”, shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion and the like.
The term “selectable marker gene” refers to a gene that when expressed confers a selectable phenotype, such as antibiotic resistance, on a transformed cell or plant. Selectable markers useful in plastid transformation vectors include, without limitation, those encoding for spectinomycin resistance, glyphosate resistance, BADH resistance, and kanamycin resistance.
The term “operably linked” means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.
The term “DNA construct” refers to a genetic sequence used to transform plants and generate progeny transgenic plants. These constructs may be administered to plants in a viral or plasmid vector. However, most preferred for use in the invention are plastid transformation vectors. Other methods of delivery such as Agrobacterium T-DNA mediated transformation and transformation using the biolistic process are also contemplated to be within the scope of the present invention. The transforming DNA may be prepared according to standard protocols such as those set forth in “Current Protocols in Molecular Biology”, eds. Frederick M. Ausubel et al., John Wiley & Sons, 1995.
As used herein, the term “chloroplast” includes organelles or plastids found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis. Chloroplasts contain chlorophyll. Chloroplasts have a higher copy number and expression levels of the transgene. Each chloroplast may contain up to 100 genomes, while each plant cell may contain up to 100 chloroplasts. Therefore, each plant cell may contain as many as 100000 chloroplast genomes which results in high expression levels of proteins expressed via the chloroplast genome. Chloroplasts further offer gene containment through maternal inheritance as the chloroplast genome is not transferred through pollen unlike nuclear genomic DNA. Chloroplasts have the ability to transcribe polycistronic RNA and can perform the correct processing of eukaryotic proteins including the ability to carry out post-translational modifications such as disulphide bonding, assembly of multimers and lipid modifications.
As used herein, a “composition,” “pharmaceutical composition” or “therapeutic agent” all include a composition comprising a myelin basic protein comprising construct as described herein. Optionally, the “composition,” “pharmaceutical composition” or “therapeutic agent” further comprises pharmaceutically acceptable diluents or carriers.
As used herein, the term “expression” in the context of a gene or polynucleotide involves the transcription of the gene or polynucleotide into RNA. The term can also, but not necessarily, involves the subsequent translation of the RNA into polypeptide chains and their assembly into proteins.
A plant remnant may include one or more molecules (such as, but not limited to, proteins and fragments thereof, minerals, nucleotides and fragments thereof, plant structural components, etc.) derived from the plant in which the protein of interest was expressed. Accordingly, a composition pertaining to whole plant material (e.g., whole or portions of plant leafs, stems, fruit, etc.) or crude plant extract would certainly contain a high concentration of plant remnants, as well as a composition comprising purified protein of interest that has one or more detectable plant remnants. In a specific embodiment, the plant remnant is rubisco.
In another embodiment, the invention pertains to an administrable composition for treating or preventing disease via administration of a therapeutic fusion protein produced in a plant chloroplast. The composition comprises a therapeutically-effective amount of the fusion protein expressed by a plant and a plant remnant.
Proteins expressed in accord with certain embodiments taught herein may be used in vivo by administration to a subject, human or animal in a variety of ways. The pharmaceutical compositions may be administered orally or parenterally, i.e., subcutaneously, intramuscularly or intravenously, though oral administration is preferred.
Oral compositions produced by embodiments of the present invention can be administered by the consumption of the foodstuff that has been manufactured with the transgenic plant producing the plastid derived therapeutic fusion protein. The edible part of the plant, or portion thereof, is used as a dietary component. The therapeutic compositions can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration. Orally, the composition can be administered in the form of tablets, capsules, granules, powders, chewable gums, and the like with at least one vehicle, e.g., starch, calcium carbonate, sucrose, lactose, gelatin, etc. The preparation may also be emulsified. The active immunogenic or therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, e.g., water, saline, dextrose, glycerol, ethanol or the like and combination thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants. In a preferred embodiment the edible plant, juice, grain, leaves, tubers, stems, seeds, roots or other plant parts of the pharmaceutical producing transgenic plant is ingested by a human or an animal thus providing a very inexpensive means of treatment of or immunization against disease.
In a specific embodiment, plant material (e.g. lettuce, tomato, carrot, low nicotine tobacco material etc,) comprising chloroplasts capable of expressing the therapeutic fusion protein, is homogenized and encapsulated. In one specific embodiment, an extract of the lettuce material is encapsulated. In an alternative embodiment, the lettuce material is powderized before encapsulation.
In alternative embodiments, the compositions may be provided with the juice of the transgenic plants for the convenience of administration. For said purpose, the plants to be transformed are preferably selected from the edible plants consisting of tomato, carrot and apple, among others, which are consumed usually in the form of juice.
According to another embodiment, the subject invention pertains to a transformed chloroplast genome that has been transformed with a vector comprising a heterologous gene that expresses a therapeutic fusion protein or peptide as disclosed herein.
Reference to the protein sequences herein relate to the known full length amino acid sequences as well as at least 12, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 265 contiguous amino acids selected from such amino acid sequences, or biologically active variants thereof. Typically, the polypeptide sequences relate to the known human versions of the sequences.
Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active therapeutic fusion polypeptide can readily be determined by assaying for native activity, as described for example, in the specific Examples, below.
Reference to genetic sequences herein refers to single- or double-stranded nucleic acid sequences and comprises a coding sequence or the complement of a coding sequence for polypeptide of interest. Degenerate nucleic acid sequences encoding polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 60, preferably about 75, 90, 96, or 98% identical to the cDNA may be used in accordance with the teachings herein polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. Complementary DNA (cDNA) molecules, species homologs, and variants of nucleic acid sequences which encode biologically active polypeptides also are useful polynucleotides.
Variants and homologs of the nucleic acid sequences described above also are useful nucleic acid sequences. Typically, homologous polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions: 2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% base pair mismatches.
Species homologs of polynucleotides referred to herein also can be identified by making suitable probes or primers and screening cDNA expression libraries. It is well known that the Tm of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Nucleotide sequences which hybridize to polynucleotides of interest, or their complements following stringent hybridization and/or wash conditions also are also useful polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., 1989, at pages 9.50-9.51.
Typically, for stringent hybridization conditions a combination of temperature and salt concentrations should be chosen that is approximately 12-20° C. below the calculated Tm of the hybrid under study. The Tm of a hybrid between a polynucleotide of interest or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962): Tm=81.5° C.-16.6 (log 10 [Na+])+0.41 (% G+C)-0.63 (% formamide)-600/1), where 1=the length of the hybrid in base pairs.
Stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C. The following materials and methods are provided to facilitate the practice of the present invention.
Codon Optimization
To maximize the expression of heterologous genes in chloroplasts, a chloroplast codon optimizer program was developed based on the codon preference of psbA genes across 133 seed plant species. All sequences were downloaded from the National Center for Biotechnology Information (NCBI, ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=2759&opt=plastid). The usage preference among synonymous codons for each amino acid was determined by analyzing a total of 46,500 codons from 133 psbA genes. The optimization algorithm (Chloroplast Optimizer v2.1) was made to facilitate changes from rare codons to codons that are frequently used in chloroplasts using JAVA.
Creation of Transplastomic Lines
The native sequence of the FVIII heavy chain (HC) was amplified using pAAV-TTR-hF8-mini plasmid (Sherman et al., 2014) as the PCR template. The codon-optimized HC sequence obtained using Codon Optimizer v2.1 was synthesized by GenScript (Piscataway, N.J., USA). We also optimized the FVIII light chain (LC), IFG-1 and mutanase. The native VP1 gene (906 bp) of Sabin 1 (provided by Dr. Konstantin Chumakov, FDA) was used as the template for PCR amplification. The codon-optimized VP1 sequence was also synthesized by GenScript. Amplified and synthetic gene sequences were cloned into chloroplast transformation vectors pLSLF and pLD-utr for Lactuca sativa and Petite Havana, respectively. Sequence-confirmed plasmids were used for bombardment to create transplastomic plants as described previously (Verma et al., 2008). Transplastomic lines were confirmed using Southern blot analysis as described previously (Verma et al., 2008) except for probe labeling and detection, for which DIG high prime DNA labeling and detection starter kit II (Roche, cat no. 11585624910) was used.
Evaluation of Translation
To compare the level of protein expression between native and codon-optimized sequences, immunoblot and densitometric assay were performed using anti-CNTB antibody. Total protein from E. coli was extracted by sonicating resuspended E. coli cells in 1×PBS and 5 mM EDTA. For total plant protein, powdered lyophilized plant cells were suspended in extraction buffer (100 mM NaCl, 10 mM EDTA, 200 mM Tris-Cl pH 8.0, 0.05% (v/v) Tween-20, 0.1% SDS, 14 mM β-ME, 400 mM sucrose, 2 mM PMSF, and proteinase inhibitor cocktail) in a ratio of 10 mg per 500 μL and incubated on ice for 1 h for rehydration. Suspended cells were sonicated (pulse on for 5 s and pulse off for 10 s, sonicator 3000, Misonix) after vortexing (˜30 s). After Bradford assay, equal amounts of homogenized proteins were loaded and separated on SDS-polyacrylamide with known amounts of CNTB protein standard. To detect CNTB fusion proteins, anti-CNTB polyclonal antibody (GenWay Biotech Inc., San Diego, Calif.) was diluted 1:10,000 in 1×PBST (0.1% Tween-20) and then membranes were probed with goat anti-rabbit IgG-HRP secondary antibody (Southern Biotechnology, 4030-05) diluted 1:4,000 in 1×PBST. Chemiluminescent signals were developed on X-ray films, which were used for quantitative analysis with Image J software (IJ 1.46r; NIH).
Evaluation of Transcripts
Total RNA was extracted from leaves of plants grown in agar medium in tissue culture room using an easy-BLUE™ total RNA extraction kit (iNtRON, cat no. 17061). For the RNA gel blot, equal amounts of total RNA (4μg) were separated on a 0.8% agarose gel (containing 1.85% formaldehyde and 1×MOPS) and blotted onto a nylon membrane (Nytran SPC; Whatman, Buckinghamshire, UK). For northern blot, The PCR-amplified product from psbA 5′ or 3′UTR region of chloroplast transformation plasmid was used as the probe. The hybridization signals on membranes were detected using DIG labeling and detection kit as described above.
Lyophilization
Confirmed homoplasmic lines were transferred to a temperature- and light-controlled greenhouse. Mature leaves from fully grown transplastomic plants were harvested and stored at −80° C. before lyophilization. To freeze-dry plant leaf materials, frozen, crumbled small leaf pieces were sublimated under 400 mTorr vacuum while increasing the chamber temperature from −40° C. to 25° C. for 3 days (Genesis 35XL, VirTis SP Scientific). Dehydrated leaves were powdered using a coffee grinder (Hamilton Beach) at maximum speed, tobacco was ground 3 times for 10 sec each and lettuce was ground 3 times for 5 sec. Powdered leaves were stored in a container under air-tight and moisture-free condition at room temperature with silica gel.
Frozen CTB-VP1 tobacco leaves were transported to a lyophilizer (Genesis 35XL, SP Scientific, Stone Ridge, N.Y.) on dry ice and lyophilized at −40° C., −30° C., −20° C., −15° C., −10° C., −5° C. and 25° C. for a total of 72 h under a 400 mTorr vacuum. Lyophilized leaf materials were ground in a coffee grinder (Hamilton Beach, Southern Pines, N.C., USA) 3 times at maximum speed (pulse on 10 s and off 30 s). The fine powder was stored with silica gel in a moisture-free environment at room temperature.
Protein Extraction and Sample Preparation for Mass Spectrometry Analysis
Total protein was extracted from 10 mg of lyophilized leaf powder by adding 1 mL extraction buffer (2% SDS, 100 mM DTT, 20 mM TEAB). Lyophilized leaf powder was incubated for 30 min at RT with sporadic vortexing to allow rehydration of plant cells. Homogenates were then incubated for 1 h at 70° C., followed by overnight incubation at RT under constant rotation. Cell wall/membrane debris was pelleted by centrifugation at 14,000 rpm (approx. 20,800 rcf). The procedure was performed in duplicate.
All protein extracts (100 μl) were enzymatically digested with 10 μg trypsin/Lys-C (Promega) on a centrifugal device with a filter cut-off of 10 kDa (Vivacon) in the presence of 0.5% sodium deoxycholate, as previously described (Leon et al., 2013). After digestion, sodium deoxycholate was removed by acid precipitation with 1% (final concentration) trifluoroacetic acid. Stable Isotope standard (SIS) peptides (>97% purity, C-term Lys and Arg as Lys U-13C6; U-15N2 and Arg U-13C6; U-15N4, JPT Peptide Technologies) were spiked into the samples prior to desalting. Samples were desalted prior to MS analysis with OligoR3 stage-tips (Applied Biosystems). The initial protein extract (10 μl) was desalted on an OligoR3 stage tip column. Desalted material was then dried on a speed vacuum device and suspended in 6 μL of 0.1% formic acid in water. MS analysis was performed in duplicate by injecting 2 μl of desalted material into the column.
PRM Mass Spectrometry Analysis and Data Analysis
Liquid chromatography-coupled targeted mass spectrometry analysis was performed by injecting the column with 2 μL of peptide, corresponding to the amount of total protein extracted and digested from 33.3 μg of lyophilized leaf powder, with 34 fmol of each SIS peptide spiked in. Peptides were separated using an Easy-nLC 1000 (Thermo Scientific) on a home-made 30 cm×75 μm i.d. C18 column (1.9 μm particle size, ReproSil, Dr. Maisch HPLC GmbH). Mobile phases consisted of an aqueous solution of 0.1% formic acid (A) and 90% acetonitrile and 0.1% formic acid (B), both HPLC grade (Fluka). Peptides were loaded on the column at 250 nL/min with an aqueous solution of 4% solvent B. Peptides were eluted by applying a non-linear gradient for 4-7-27-36-65-80% B in 2-50-10-10-5 min, respectively.
MS analysis was performed using the parallel reaction monitoring (PRM) mode on a Qexactive mass spectrometer (Thermo Scientific) equipped with a nanospray Flex™ ion source (Gallien et al., 2012). Isolation of targets from the inclusion list with a 2 m/z window, a resolution of 35,000 (at m/z 200), a target AGC value of 1×106, and a maximum filling time of 120 ms. Normalized collision energy was set at 29. Retention time schedules were determined by the analysis of SIS peptides under equal nanoLC chromatography. A list of target precursor ions and retention time schedule is reported in the Supplementary Information. PRM data analysis was performed using Skyline software (MacLean et al., 2010).
Ribosome Profiling
Second and third leaves from the top of the plant were harvested for ribosome profiling. Lettuce plants were approximately 2 months old. Tobacco plants were 2.5 or 2 months old, for native and codon-optimized VP1 constructs, respectively. Leaves were harvested at noon and flash frozen in liquid nitrogen. Ribosome footprints were prepared as described in Zoschke et al (2013) except that ribonuclease I was substituted for micrococcal nuclease. Ribosome footprints were converted to a sequencing library with the NEXTflex Illumina Small RNA Sequencing Kit v2 (BIOO Scientific, 5132-03). rRNA contaminants were depleted by subtractive hybridization after first strand cDNA synthesis using biotinylated oligonucleotides corresponding to abundant rRNA contaminants observed in pilot experiments. Samples were sequenced at the University of Oregon Genomics Core Facility. Sequence reads were processed with cutadapt to remove adapter sequences and bowtie2 with default parameters to align reads to the engineered chloroplast genome sequence.
Chloroplast Vector Construction and Regeneration of Transplastomic Plants
The native VP1 gene (906 bp) of Sabin type 1 poliovirus (provided by Dr. Konstantin Chumakov, FDA) was amplified using forward primer 5′-gggCCCgggCCCCggCgTAAACgCTCTgTTgggTTAggTCAgATg-3′ (SEQ ID NO: 11) and reverse primer 5′-CgATCTAgATCAATATgTggTCAgATC-3′ (SEQ ID NO: 12). The PCR-amplified fragment and the codon-optimized VP1 gene (synthesized by GenScript, Piscataway, N.J., USA) were cloned into tobacco and lettuce chloroplast transformation vectors. Biolistic delivery of chloroplast transformation vectors and regeneration of transplastomic tobacco (Nicotiana tabacum cv. Petit Havana) and lettuce (Lactuca sativa cv. Simpson Elite) lines were performed as previously described (Ruhlman et al., 2007; Verma et al., 2008).
Characterization of Transplastomic Tobacco and Lettuce Lines
To confirm transgene cassette integration into the chloroplast genome, PCR was performed using primer pairs 3P/3M and 5P/2M or 16S-Fw/3M and 5P/2M for tobacco and lettuce, respectively (Verma et al., 2008; Kanagaraj et al., 2011). Southern blot analysis was performed to confirm transgene integration and homoplasmy as previously described (Verma et al., 2008).
Immunoblot Analysis and Purification of Chloroplast-Derived Proteins
Immunoblot analysis and quantitation of CTB-VP1 fusion proteins were performed according to previously published methods (Davoodi-Semiromi et al., 2010). To detect CTB-VP1-fused proteins, blots were incubated with 1:10,000 rabbit anti-CTB polyclonal antibody (GeneWay, San Diego, Calif., USA) or 1:1,000 rabbit anti-VP1 polyclonal antibody (Alpha Diagnostic Intl. Inc., San Antonio, Tex., USA) followed by 1:4,000 goat anti rabbit IgG-HRP as secondary antibody (SouthernBiotech, Birmingham, Ala., USA). CTB (Sigma, St Louis, Mo., USA) and recombinant Sabin 1 VP1 (Alpha Diagnostic Intl. Inc., San Antonio, Tex., USA) were used as positive controls. To purify chloroplast-derived CTB-VP1 fusion proteins, His60 Ni Superflow Resin (Clontech Laboratories, Mountain View, Calif., USA) was used according to the manufacturer's instructions. Eluted fractions were dialyzed 3 times with sterile phosphate-buffered saline (PBS), aliquoted and stored at −20° C. Purified chloroplast-derived CTB-VP1 was used for immunoglobulin measurements.
Cholera Toxin-B-GM1-Ganglioside Receptor Binding Assay
To test the ability of the tobacco chloroplast-derived CTB-VP1 to form pentamers and bind to the GM1-ganglioside receptor, a CTB-GM1 binding assay was performed as described (Davoodi-Semiromi et al., 2010).
Mice and Immunization Schedule
Female CD-1 mice aged 6-7 weeks were purchased from Charles River Laboratories (Wilmington, Mass., USA) and housed in microisolator cages. Experiments were conducted in accordance with guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee. Mice were randomly divided into 9 groups of 10 mice per group. Group 1 was a control group in which mice were untreated. All mice from groups 2 through 8 were subcutaneously (s.c.) primed with 100 μl of IPV suspension of three types of poliovirus (Type 1 (Mahoney), Type 2 (MEF-1), and Type 3 (Saukett) (IPOL, Sanofi Pasteur SA)). Group 2 mice were s.c. boosted with the same IPV 30 days after priming. Mice in groups 3 through 9 were orally boosted with lyophilized plant material: mice in groups 3-8 were boosted once a week for 8 consecutive weeks starting one week after priming. Mice in groups 3 through 5 were orally boosted with lyophilized native CTB-VP1-expressing leaves; each mouse was boosted with 20 mg of material in 200 μl of PBS plus different adjuvants: saponin (group 3), squalene (group 4) or both (group 5). Mice in groups 6 through 9 were orally boosted with lyophilized codon-optimized CTB-VP1-expressing leaves; each mouse was boosted with 20 mg of material in 200 μl of PBS plus different adjuvants: saponin (group 6), squalene (group 7) or both (groups 8 and 9). Blood was collected one day prior to priming and 7 days after boosting. Serum samples were heat-inactivated at 56° C. for 30 min to destroy complement activity.
Preparation of Vaccine Formulations of Bioencapsulated, Plant-Made CTB-VP1 Protein
Vaccine formulation was generally performed as previously described39, 40 but with modifications. Briefly, the double emulsion technique was used to prepare vaccine formulations. To prepare VP1 antigen adjuvanted with squalene, the primary emulsion in the aqueous phase was made by mixing 0.05% Tween-80 in PBS with 20 mg of lyophilized VP1 antigen. The oil phase was a combination of squalene (80% v/v) and Span-80 (Sigma, P4780) (20% v/v). The emulsion was made by mixing the primary oil emulsion with the aqueous phase and adjusting the total volume to 200 μl per mouse with PBS, then homogenizing at 5,000 rpm for 5 min.
To prepare VP1 antigen adjuvanted with saponin, 20 mg lyophilized native or codon-optimized CTB-VP1 plant material was mixed with 2 mg saponin per mouse and PBS was added to a final volume of 200 μl. To prepare VP1 antigen with both adjuvants, a primary emulsion was made by mixing 20 mg of lyophilized native or codon-optimized VP1 with 2 mg saponin in PBS containing 0.05% Tween-80. Then, the primary emulsion was then gently mixed with a squalene emulsion containing squalene (80% v/v) and Span-80 (20% v/v).
Determination of Antibody Response by ELISA
Immunological responses, including serum levels of VP1-specific IgG1 and IgA titers, were assayed by direct ELISA and in vitro poliovirus Sabin 1, 2 and 3 neutralizing assays, which were performed by the Centers for Disease Control and Prevention (CDC). Briefly, for the antibody response assay, 10 μg/ml purified CTB-VP1 protein was used to coat 96-well Maxisorp ELISA plates (Nunc) overnight at 4° C. Plates were blocked with 1% BSA (Sigma 7906) in PBS with 0.05% Tween. Starting with a 1:400 dilution, two-fold dilutions of individual heat-inactivated sera samples were incubated overnight at 4° C. Secondary antibodies were HRP-conjugated rat anti-mouse IgG1 (BD Pharmingen, 559626, 1:1,000) and HRP-conjugated goat anti-mouse IgA (American Qualex, A138N, 1:5,000) diluted in blocking buffer and incubated at 37° C. for 1 h followed by color development with TMB substrate (ES001, EMD Millipore, CA) for 10 min at RT. The reaction was stopped by adding 100 μl of 2N sulfuric acid to each well, and absorbance was measured using an ELISA reader at 450 nm. Antibody titers were defined as the reciprocal of the highest dilution above the cut-off, which was three times the mean background41. All sera samples were tested in triplicate. Results are shown as individual antibody titer±SEM.
Poliovirus Sabin 1, 2, 3 Neutralization Assay
After the 10th oral boost with native or codon-optimized CTB-VP1 proteins adjuvanted with saponin and/or squalene or, for group 2, both priming and boosting with the IPV, serum samples were collected and saved at −80° C. for further neutralization assays at the CDC as previously described21, 42. Briefly, sera samples were tested in triplicate with the use of modified microneutralization assays for antibodies to Sabin strains type 1, 2, and 3. Serum samples from control and experimental groups were tested randomly and blindly. The serum dilution of a reciprocal titer at which no virus neutralization was detected was recorded as the log 2 (titer) of 2.5, or negative; a log 2 titer of 3 was considered protective. Individual titers for each mouse are plotted and the bar represents mean neutralizing titer±SEM.
Statistical Analysis
All data are reported for individual mice and mean±SEM is given for each group. Analyses for statistically significant differences in antibody titers between groups were performed using Student's t-test (GraphPad Prism version 6) and P values<0.05 were considered significant.
The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.
Codon-Optimization of Human/Viral Transgenes
The differences in codon usage by chloroplasts are often associated with a decrease in translation. In efforts to increase expression of therapeutically relevant proteins, the native sequences of blood clotting factor VIII heavy chain (FVIII HC and FVIII LC), and IGF-1, from human, viral capsid protein 1 (VP1) from polio virus and mutanase from bacteria were analyzed as previous studies showed very low expression levels of <0.005% for FVIII and ˜0.1% for VP1, for example. Codon optimization software was developed to increase translation using an algorithm based on the analysis of codons used in psbA genes from 133 plant species because the psbA gene is most highly expressed gene in chloroplasts (
Evaluation of Translation of Native and Synthetic Genes in E. coli
In this study, the native sequences for FVIII HC (2262 bp), FVIII LC and FVIIISCc (a fusion form of codon-optimized FVIII heavy chain (HC including 14 amino acids from B domain) and light chain (LC) (
Before creation of transplastomic plants expressing the codon-optimized CNTB-FVIII HC and CNTB-VP1, the synthetic genes were first transformed into E. coli to evaluate their expression. Because of prokaryotic origin, chloroplasts have similar transcription/translation machinery. As seen in
Translation Efficiency of Native and Codon-Optimized Genes in Lettuce and Tobacco Chloroplasts
After confirmation of improvement in expression of synthetic sequences in E. coli, the transformation vectors containing synthetic FVIII HC and VP1 sequence were used to create transplastomic lettuce and tobacco plants expressing codon-optimized HC and VP1. To confirm homoplasmy, Southern blot analysis was performed with four independent lettuce and tobacco lines expressing native and codon-optimized FVIII HC, and lines expressing native and codon-optimized VP1. For lettuce plants expressing CNTB-FVIII HC, native and codon-optimized sequence, chloroplast genomic DNA was digested by HindIII and probed with dig-labelled probe spanning flanking region (
Expression levels of codon-optimized or native gene sequences were quantified using immunoblot and densitometry assays (
To investigate the impact of codon optimization on transcript stability, northern blots were performed with a probe, psbA 5′ or 3′ UTR sequence (
Absolute Quantitation by PRM Analysis
Expression levels of codon-optimized and native gene sequences were also quantified using PRM mass spectrometry (
The content of VP1 protein of codon-optimized plant was calculated as results of PRM measurement of the three CNTB tryptic peptides (
Absolute quantitation can be achieved by spiking a known amount of the counterpart SIS peptide into the samples. For each counterpart SIS peptide (34 fmol) was injected on column mixed with protein digest (equivalent to protein extracted from 33.3 μg lyophilized leaf powder). By calculating ratios of area under the curve (AUC) of each, SIS and endogenous peptides, we estimated the endogenous peptide molarity, expressed as femtomole on column (
Ribosome Profiling Studies
Ribosome profiling uses deep sequencing to map “ribosome footprints”—the mRNA fragments that are protected by ribosomes from exogenous nuclease attack. The method provides a genome-wide, high resolution, and quantitative snapshot of mRNA segments occupied by ribosomes in vivo (Ingolia et al., 2009). Overall ribosome footprint coverage can provide an estimate of translational output, and positions at which ribosomes slow or stall are marked by regions of particularly high ribosome occupancy.
To examine how codon optimization influenced ribosome behavior, we profiled ribosomes from plants expressing the native and codon optimized CNTB-FVIII HC and CNTB-VP1 transgenes.
The number of ribosome footprints in FVIII line decreased ˜2-fold in the codon-optimized line, whereas protein accumulation increased 5-7 fold. However, a major ribosome pause can be observed near the 3′ end of the native transgene, followed by a region of very low ribosome occupancy (see bracketed region in
Codon Usage is not Similar Between E. coli and Chloroplasts
The codon-optimized FVIII HC, LC and SC sequences improved expression level in E. coli 7-10 fold. Homoplasmic lines (transformation of all chloroplast genomes) were confirmed by Southern blot. The highest levels of expression level of codon-optimized CTB-FVIII heavy chain (100 kDa), light chain (92 kDa) and single chain (179 kDa) were 2440, 160 and 230 μg/g in lyophilized plant cells, respectively. In single chain lettuce, the expression level increased from 150 μg/g to 230 μg/g with age from 26-day to 48-day. Translation efficiency of synthetic genes was first tested in E. coli expression system because of the prokaryotic origin of chloroplasts. However, the expression of the synthetic VP1 gene showed only 3 times higher than the native gene. Lower level of translation of synthetic VP1 than FVIII HC could be due to differences in rarely used codons between E. coli and chloroplast. In E. coli, among 6 arginine codons, four (AGG, AGA, CGG and CGA) codons are not preferred. Also, GGA for glycine, AUA for isoleucine, CUA for leucine, and CCC for proline (Kane, 1995) are least preferred codons in E. coli. Usually the small number of rare codons do not cause severe impediment in translation. However, clustering of a large number of the rare codons affects translation. Arginine codons AGG/AGA have been extensively studied for their detrimental effect on protein expression in E. coli. In a study which used a test protein with different magnitude of tandem repeat of AGG codons, translation was drastically reduced when the number of AGG clusters was two to five (Rosenberg et al., 1993). While there is no clustering issue of the rare codons for the native VP1, it was found that the native sequence of FVIII HC has 3 clustering sites where rare codons for arginine or glycine were consecutively placed, for example, at codons 3 and 4 (AGA-AGA), codons 489 and 499 (AGG-AGA), and codons 562 and 563 (AGA-GGA). So the elimination of the multiple repeat of rare Arg codons from native FVIII HC sequence by codon optimization could increase translation of the synthetic HC in E. coli. In contrast, the native sequence of VP1 has no such tandem repeats of the rare codons so that the expression efficiency was less affected than FVIII HC native sequence. In view of these data it is clear that production and oral delivery of FVIII SC clotting factor using edible lettuce will benefit patients with increased compliance, in a cost-effective and safe manner. Large scale/clinical grade production of therapeutic plant leaves at cGMP facility will reinforce evaluation of plant-made clotting factors in large animal models, non-human primates and facilitate toxicology studies.
Codon Optimization Significantly Enhances Translation in Chloroplasts
The increase of 22.5˜28.0 fold (by PRM) and 46-48 fold (by WB) between the native and codon-optimized VP1 in chloroplasts is quite remarkable. Since the codon optimizer was designed to optimize expression of heterologous genes in chloroplasts, it is expected that improvement of expression level between native and synthetic sequence in chloroplasts is much greater than that of expression in E. coli. For example, CUA for leucine is rarely used in E. coli but the same codon is most favorably used in chloroplasts. The codon optimization program increased the ratio of CUA among 6 leucine codons from 27.8% of native sequence to 38.9% of codon-optimized sequence for VP1. In contrast to expression in E. coli, fold difference of protein level between VP1 plants expressing native and codon-optimized sequence was greater than that of plants expressing FVIII HC between native and codon-optimized sequence. Given that higher molecular weight of FVIII (754 amino acids) than VP1 (302 amino acids) requiring more tRNAs and amino acids in chloroplasts, the resultant protein synthesis will be less efficient. Considering that chloroplasts have an extremely high capacity to synthesize and accumulate foreign proteins, the nitrogen supply and amino acid pool could be a major concern for accumulation of recombinant proteins. As seen in previous report (Bally et al., 2009), total amino acid content of transplastomic plants was significantly affected with reduction of resident proteins, especially Rubisco, due to the limited resources of protein synthesis, which usually functions as a major leaf amino acid storage protein.
Codon usage in psbA (our program) is different for preferred Arg, Asn, Gly, His, Leu and Phe codons than those reported for 79 tobacco chloroplast mRNAs based on in vitro studies (Nakamura and Sugiura, 2007). Preferred codons are decoded more rapidly than non-preferred codons, presumably due to higher concentrations of the corresponding tRNAs that recognize the preferred codons, which speed up elongation rate of protein synthesis (Yu et al., 2015). Higher plant chloroplast genomes code for a conserved set of 30 tRNAs. This set is believed to be sufficient to support translation machinery in chloroplast (Lung et al., 2006). In the ribosome profiling data for codon optimized VP1, two major peaks representing presumed sites of ribosome stalling correlated with an unusually high concentration of serine codons (
In previous studies, codon modification to improve expression level of heterologous genes was focused on the increase of AT content by changing third nucleotide of codons. In case of IGF-1 (Daniell et al., 2009), the synthesized sequence of IGF-1 changed by 3rd position of codons showed the dramatic fold increase of expression over the native sequence in E. coli system but no increase of expression level was observed in chloroplasts, suggesting that increase of AT content is not the major contributing factor in enhancing translation. As seen in this study, the AT content of codon optimized VP1 was marginally increased but the protein level of the optimized CTB-VP1 was dramatically increased up to 22.56˜28.0 fold (by PRM) and 46-48 fold (by WB) over native sequence when expressed in chloroplasts. Therefore, several other factors play a key role in regulating efficiency of translation. As observed in ribosome profiling studies of CNTB-VP1, the availability and density of specific codons could severely impact translation. Similarly, FVIII HC, ribosome footprint results showed that ribosome pause was mapped to CTC leucine codons which are almost not used in psbA genes. The codon is also rarely used in lettuce rbcL gene (2.44%) and for tobacco rbcL, the codon is never used. Native FVIII HC uses the CTC codon as high as 15.28% but CTC codon was eliminated from the codon-optimized sequence according to psbA codon usage. More detailed analysis of codon frequency of the native FVIII HC and the psbA gene reveals further insight into rare codons; GGG for Gly is used 2.3% in psbA but 11.63% in HC native; CTG for Leu is 3.7% in psbA but 26.39% in HC native; CCC for Pro is 1.9% vs 11.9%; CGG for Arg is 0.5% vs 10.81%; CTG for Val is 1.7% vs 25.49%. So, similar to CTC codon, several other rare codons described above in the native human gene should have decreased translational efficiency in chloroplasts.
New Solution for Quantitation of Insoluble Multimeric Proteins
A major challenge is the lack of reliable methods to quantify insoluble proteins because the only reliable method (ELISA) can't be used due to aggregation or formation of multimeric structures. However, delivering accurate doses of protein drugs is a fundamental requirement for their clinical use. Therefore, in this study we carried out parallel reaction monitoring (PRM) analysis for absolute quantitation of CNTB-FVIII HC and CNTB-VP1 in plants carrying codon optimized and native sequences. PRM analysis has been broadly adopted in quantitative proteomics studies, e.g. biomarker discovery in plasma, due to its high sensitivity, specificity and precise quantitation of specific protein targets whiting complex protein matrices (Gallien et al., 2012). These qualities clearly show the advantage of using PRM in the quantification of specific protein targets, independently of the protein matrix source (e.g. plant extracts from tobacco or lettuce) or complexity. Moreover, the development of a PRM assay for hand full of proteins can be achieved in a relatively short time and at low costs (not taken in consideration the MS instrumentation). As a peptide-centric quantitation methodology also offers robustness and versatility of protein extraction methods and keeping the protein of interest in a native conformation is not required. However, it is intrinsically biased by the enzymatic cleavage site access of the enzymes used for digestion. In order to overcome this bias, we have used strong denaturing conditions (i.e. 2% SDS) and buffers that favor activity of the proteolytic enzymes (i.e. sodium deoxycholate based buffers) (Leon et al., 2013). For FVIII HC (
Heterologous gene expression utilizing chloroplast genome sequences, ribosome profiling and targeted mass spectrometry (MS) was analyzed to enhance our understanding of synthesis of valuable biopharmaceuticals in chloroplasts. Targeted Proteomic Quantification by Mass Spectrometry showed that codon optimization increases translation efficiency 5-50 fold based on the coding sequence, validating this approach for the first time for quantitation of protein drug dosage in plant cells. The lack of reliable methods to quantify insoluble proteins due to aggregation or formation of multimeric structures is a major challenge. Both biopharmaceuticals used in this study are CNTB fusion proteins that form pentamers, which is a requirement for their binding to intestinal epithelial GM1 receptors. Such a multimeric structure excluded the commonly used ELISA for quantitation of dosage. However, delivering accurate doses of protein drugs is a fundamental requirement for their clinical use and this important goal was accomplished in this study. Indeed plant biomass generated in this study has resulted in development of a polio booster vaccine, validated by the Center for Disease Control, a timely invention to meet World Health Organization requirement to withdraw current oral polio vaccine in April 2016, that cause severe polio in outbreak areas.
Such increase of codon-optimized protein accumulation is at the translational level rather than any impact on transcript abundance or stability. The codon-optimizer program increases transgene expression in chloroplasts in both tobacco and lettuce, with no species specificity. In contrast to previous in vitro studies, first in depth in vivo studies of heterologous gene expression using a wealth of newly sequenced chloroplast genomes facilitated the development of a new codon optimizer program which was tested using two important proteins for clinical applications. Ribosome foot prints obtained using profiling studies did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but it is a valuable tool for diagnosing rate limiting steps in translation. A major ribosome pause at CTC leucine codons, a rarely used codon in chloroplasts was eliminated from the native gene after codon optimization. Ribosome stalls observed at clusters of other codons in the codon-optimized genes provide opportunity for further optimization by eliminating the codons that cause such stalls.
Construction of Plant Transformation Vectors
Two VP1 proteins derived from Sabin 1 coding sequences (CDS) were expressed in tobacco and lettuce chloroplasts. See
Integration of Foreign Genes into Tobacco and Lettuce Plastomes
CTB-VP1 transplastomic lines were generated by biolistic particle bombardment. After selection on spectinomycin-containing media, putative transplastomic lines were confirmed by PCR analysis with primer sets 3P/3M and 5P/2M for tobacco or 16S-Fw/3M and 5P/2M for lettuce (data not shown). Targeted integration and homoplasmy of the CTB-VP1 gene was further verified by Southern blot probed with the trnI and trnA flanking sequence (
Folding, Stability and CTB-VP1 Pentamer Assembly in Lyophilized Tobacco Leaves
CTB-VP1 accumulation in transplastomic plants was quantified by western blot analysis. Intensities of CTB-VP1 protein in the bands in native and codon-optimized plants were compared with known amounts of CTB standard. The western blot analysis indicated that the codon-optimized VP1 sequence significantly increased accumulation of CTB-VP1 when compared with the native VP1 gene product. Native and codon-optimized CTB-VP1 reached up to 0.1% and 4-5% of the total leaf protein, respectively (up to 100-fold increase based on quantitation using targeted MS or western blots, data not shown). As shown in
Animal Vaccination and Antibody Responses to VP1
Plant-derived subunit vaccines are heat-stable and are free from contamination with animal pathogens. They can also be engineered to contain multiple antigens and transmucosal carrires, to protect against multiple infectious diseases. Such mechanistic and conceptual advances could revolutionize vaccine delivery by eliminating the cost of complex production systems, such as fermentation, purification, cold storage and transportation. Two major challenges to plant based vaccine production include the low levels of expression of antigens via the nuclear genome and the potential to induce tolerance without injectable priming of antigens with adjuvants.
In order to address inadequacies of the current OPV, including poor vaccine efficacy, instability and reversion to neuro-virulence, shedding of circulating vaccine-derived polio viruses, and the high cost and inadequate mucosal immunity of inactivated poliovirus vaccine (IPV), a low-cost booster vaccine has been developed in this study using polio viral antigen bioencapsulated in plant cells. The strategy of using a plant-made viral protein 1 (VP1) subunit vaccine for an oral booster rather than repeated OPV vaccination is a novel approach to achieve the goal of global PV eradication. In this study, we provide evidence that oral boosting with chloroplast-derived VP1 together with plant-made adjuvants (saponin and squalene) induces strong immune responses that confer protective immunity against different PV serotypes.
In the previous example, we describe lyophilized CTB-VP1 protein. In the present example, this protein was formulated with plant-derived adjuvants (saponin and/or squalene) which induce specific antibody immunogenicity and neutralize different polio virus serotypes. Mice were divided into groups as described in the Methods section and in the Table below.
Construction of Plant Transformation Vectors
Two VP1 proteins derived from Sabin 1 coding sequences (CDS) were expressed in tobacco chloroplasts. The first sequence encompassed the native 906-bp VP1 sequence (51.98% AT) fused with the transmucosal carrier CTB. The second was codon-optimized for expression in tobacco and lettuce chloroplasts. Of the 302 amino acids in the protein, 187 codons were optimized by changing the codon usage frequency to resemble that of the chloroplast psbA gene (the most highly translated chloroplast gene). Rare codons were replaced with optimal codons for transgene expression in chloroplasts and the AT content of the optimized VP1 gene increased from 51.98% to 59.03%. Both CTB-VP1 fusion genes were constructed with a GPGP (Gly-Pro-Gly-Pro) hinge region to minimize steric hindrance of the fused VP1, as well as a furin cleavage site, RRKRSV (Arg-Arg-Lys-Arg-Ser-Val) (SEQ ID NO: 14) (
Animal Vaccination and Antibody Responses to VP1
As mentioned above, mice were divided into groups as set forth in the table above. One day prior to immunization, mice from all groups were bled. We determined serum titers of VP1-specific IgG1 and IgA antibodies at various time points on days 29, 43, 57, 87 and 117 after boosting with IPV or CTB-VP1 with adjuvants. At all tested time points, systemic and mucosal immune responses were quantified with ELISA. VP1-IgG1 titers reached highest levels in the first month and remained at the same level. Further boosting did not increase VP1-IgG1 levels (See
Furthermore, vaccination with codon-optimized VP1 induced significantly higher serum titers of specific anti-VP1 IgG1 and IgA, showing that high expression of antigen in lyophilized codon-optimized material is critical for effective immunization. Moreover, boosting with either native or codon-optimized VP1 antigens combined with two adjuvants (groups 5, 8 and 9) induced stronger IgG1 and IgA immune responses than with either one adjuvant, suggesting that plant-derived adjuvants enhance delivering antigens via the mucosal rote and developing strong mucosal and systemic immune responses.
Poliovirus Neutralizing Titers Against all Sabin 1, 2 and 3 Strains Following Priming and Boosting
To determine if anti-VP1 IgG1 and IgA antibodies can neutralize poliovirus, virus neutralization titers were measured for all three Sabin serotypes. Blood samples from all experimental and untreated groups were tested in a double blind manner and in triplicate samples at CDC. A serum sample was considered seropositive if antibodies were present at a log2 titer≥2.5. Individual neutralization titers were plotted, and the bar represents the mean neutralizing titer±SEM of each group. Results show that after IPV priming, all experimental groups—oral boosting with native (groups 3-5) or codon-optimized VP1 antigen plus either one or both adjuvants (groups 6-9), as well as priming and boosting only with the same IPV (group 2) induced significantly higher neutralizing titers against all three Sabin strain serotypes. Results show that oral boosting with codon-optimized VP1 plus saponin and squalene (group 8) produced the most Sabin 1, Sabin 2 and Sabin 3 neutralizing antibodies, similar to the group of mice that were both primed and boosted with IPV (group 2) (
To determine the seropositivity rate of poliovirus-neutralizing antibodies, for each Sabin strain, the number of mice with seroprevalence (neutralizing antibody log2 (titer)≥3) was compared with the total number of mice in each group. Mice boosted with IPV (group 2) or orally boosted with codon-optimized VP1 antigen with saponin and squalene adjuvants (group 8) showed high seropositivity for poliovirus Sabin 1, 2 and 3 neutralizing antibodies (
After the outbreak of VDPV2, several critical global policies and processes were adopted in 2013 to support the introduction of at least one dose of IPV into routine immunization schedules to mitigate risks of withdrawal of serotype 2 OPV. The WHO's Strategic Advisory Group of Experts (SAGE) recommended the withdrawal of OPV2 from routine immunization programmes in all countries, facilitated by the introduction of at least one dose of IPV in all OPV-using countries in 2015 and the withdrawal of OPV2 globally in 2016 (the global polio eradication initiative (GPEI), 2015). To accomplish these current priorities, emphasis should be placed on needed activities including licensure and increased availability of bivalent OPV for routine immunization, as well as solid implementation of at least one dose of IPV for all OPV-using countries. However, multiple risks still remain in preparation for the global introduction of IPV and the upcoming switch from trivalent OPV (tOPV) to bivalent OPV (bOPV), including tight IPV supply, persistent cVDPV transmission and challenges to meet containment requirements (GPEI Polio Eradication & Endgame Midterm Review, 2015). Most importantly, there is no booster technology available except IPV which is not affordable for most developing countries. Further, the routine use of OPV vaccination must be discontinued for the global PV eradication, and global introduction of IPV instead of OPV is needed. At the same time, high levels of population immunity against the emergence of VDPV and future outbreaks of wild PV need to be maintained. However, the current cost per vaccine dose of IPV is too high for developing countries.
Expression of VP1 in chloroplasts and bioencapsulation in plant cells can protect antigens from the digestive system upon oral delivery and facilitates their release into the immune system in the gut by commensal microbes22,23. CTB-antigen fusions facilitate transmucosal delivery to the immune system via the GM1 intestinal epithelial receptor24. Further, CTB-fused vaccine antigens stimulate production of antigen-specific IgG and IgA after priming and oral boosters, conferring protection against toxin/pathogen challenge22. Production of green vaccines against infectious diseases with ease of oral administration that does not require a cold chain is an important need, especially in areas with limited access to cold storage and transportation22. Previous studies have demonstrated that biopharmaceutical or antigen proteins can be stored in lyophilized plant material at room temperature for several months or even 2 years without any detectable degradation25-27. VP1 is highly stable in lyophilized plant cells when stored at ambient temperature for several months.
Antigen-specific IgG and IgA were significantly induced after few oral boosts are adequate to generate high levels of systemic and mucosal immunity. Both VP1-IgG1 and VP1-IgA titers reached highest levels after the first month of oral boosting and did not increase further with more number of boosters. Although neutralization data from later stage sera collection is provided here, previous batches evaluated for Sabin serotype 1 neutralization showed similar results in groups boosted with plant cells expressing VP1 (data not shown). In this study, plant cells were suspended in PBS before oral delivery but for delivery to children, suitable formulation with sugar syrup can be required. Although IPV is highly effective in inducing systemic antibodies to protect against paralytic disease, it is less efficient in inducing the mucosal immunity that is needed to prevent re-infection and excretion of polioviruses into the environment. Our results confirmed that in mice s.c. primed/boosted with IPV minimal IgA titers, explaining the inadequate mucosal immunity of IPV. Mice s.c. primed with IPV and orally boosted with bioencapsulated VP1 elicited strong antigen-specific serum IgG1 (>12,800 titer) and IgA (>800 titer) responses, confirming that oral delivery of VP1 antigen with adjuvants generated both systemic and mucosal immune responses. Unlike viral immunization, subunit vaccines mainly induce Th2 response with IgG1 isotype antibody28-30. Oral boosting with subunit vaccines elicits both the mucosal and systemic immune response, as shown by high IgG1/IgA titers.
In this study, we evaluated both native and codon-optimized VP1 antigens expressed in chloroplasts. The level of VP1 protein was much higher in plants expressing codon-optimized VP1. Our in vivo study also showed that vaccination with codon-optimized VP1 induced much higher IgG1 and IgA antibody responses (
Neutralizing antibody levels at a titer above the 1:8 dilution (3 log2(titer)) threshold are accepted by all national regulatory agencies as having a good correlation with protection when reviewing license applications for IPV-containing vaccines21, 31. As expected, virus-neutralizing titers induced by priming and boosting with IPV were high for all Sabin strains. In our study, priming with IPV and orally boosting with bioencapsulated VP1 with adjuvant (saponin and squalene) showed the highest seropositivity and virus neutralizing titers (range 3.17-10.17 log2 titer) against all Sabin 1, 2, 3 strains. Although mice that were only boosted with VP1 plus two adjuvants but not primed (group 9) showed the strongest VP1-specific antibody (IgG1 and IgA) production, no neutralizing virus titers were observed in this group when compared to the mice that were primed with IPV. Thus, the only oral boosting with subunit vaccination appears to be insufficient to induce a good neutralizing antibody response to the antigen. These high anti-VP1 antibodies by ELISA might only bind to viral protein which are expressed on the surface of infected cells but not, to a significant degree, on free virus particles, so that they cannot neutralize virus and protect cells against viral infection32. The high concentrations of neutralizing antibody are required to inhibit cell-cell transmission of virus infection33-35. These results demonstrate that oral priming is essential to induce adequate immunity against pathogens.
Although this study focuses on polio booster vaccine, there is greater need to boost immunity as life expectancy is on the rise. Loss of immunity against infectious diseases among elderly population is a growing concern. For example, shingles occur when latent chickenpox virus is reactivated when aging weakens the immune system and this is rarely observed due to new viral infections. Therefore, to enhance immunity against a number of infectious diseases among elderly population, low cost oral booster vaccines could serve this purpose. In conclusion, virus- and cold chain-free vaccines are not currently available for any infectious disease. Therefore, production and oral delivery of vaccines using transplastomic technology will facilitate the development of low-cost cold chain- and virus-free booster vaccines. Here we show a low-cost booster vaccine using bioencapsulated polio antigens as an alternative strategy to avoid repeated OPV vaccinations for global PV eradication and the prevention of polio outbreaks in endemic areas.
Human insulin-like growth factor 1 (IGF-1) plays important roles in growth and development of skeletal muscle in myoblast/fiber formation, differentiation and regeneration after injury. Because E peptide enhances efficacy of IGF-1, it is desirable to express Pro-IGF-1 in chloroplasts to reduce cost and facilitate oral delivery.
Pro-IGF-1E (105 aa) was codon-optimized using a software developed described in Example I based the most highly expressed chloroplast gene from 133 plant species (See
Phosphorylation of IGF-1 receptor (IGFR) by plant derived CTB-Pro-IGF-1 was examined in vitro and the results are shown in
Among 105 aa, 73 codons were modified resulting in 57% AT content in codon optimized IGF-1 gene. To avoid glycosylation Lsy68, Arg74 and Arg77 were changed to Gly68, Ala74 and Ala77. See
Pro-IGF-1 increased 3-fold in blood after oral delivery of lyophilized plant cells at 8 hrs and was maintained up to 24 hrs (
The phosphorylation of IGFR by plant derived CTB-Pro-IGF-1 and the maintenance Pro-IGF-1 in the circulatory system and in the muscle tissue after oral gavage confirms suitability of this system for low cost production and delivery of functional IGF1 bioencapsulated in plant cells. Lyophilized plant cells can be stored indefinitely at ambient temperature without decrease in efficacy of IGF-1.
Expression of Pro-IGF-1 with E peptide in chloroplasts provides an effective, efficient and affordable oral drug delivery concept for treatment of disorders caused by IGF-1 deficiency including muscle disorders. This approach offers a technological breakthrough to address the rising cost of healthcare in addition to increasing patient compliance for repetitive long-term drug delivery.
Dental Caries is a prevalent biofilm-associated oral disease worldwide. Antimicrobials are minimally effective as they do not penetrate the exopolysaccharide (EPS) matrix. Therefore, in the present example, we express EPS degrading enzymes dextranase and mutanase fused with antimicrobial peptides (AMP). The recombinant enzyme production in plant chloroplasts is 1000-3,100-fold cheaper because it eliminates prohibitively expensive fermentation, purification, cold storage/transportation and invasive surgical delivery and facilitates storage at ambient temperature. The primary goal of this example is to develop chewing gums impregnated with lyophilized plant cells expressing AMP and enzymes. Hence, an initial study was performed to optimize the chewing rates and time for maximum drug release using chewing simulator with chewing gum made from lyophilized plant cells expressing reporter gene GFP.
Dextranase gene from Streptococcus nutans and mutanase gene from Paenibacillus was either fused with PG1 or without PG1 was cloned into chloroplast vectors and their functionality was evaluated first in E. coli. See
GFP in gum tablets was not significantly degraded during gum preparation. The chewing rate, time required for maximum protein release is currently optimized using chewing simulator. Accordingly, production of EPS-degrading enzymes fused with AMP should provide a promising treatment for dental caries, preferably administered as gum tablets.
While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention by limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.
This Application is a §371 of International Patent Application No. PCT/US2017/023263, filed March 20, 2017, which claims the benefit of U.S. Provisional Application No. 62,310,788, filed Mar. 20, 2016. The entire disclosure of each of the aforesaid applications is incorporated by reference in the present application.
This invention was made with government support under grant numbers HL107904, HL109442, EY024564 awarded by the National Institutes of Health and grant number 1339130 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/023263 | 3/20/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/165320 | 9/28/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20060253935 | Daniell | Nov 2006 | A1 |
| 20080038193 | Wouters et al. | Feb 2008 | A1 |
| 20110179530 | Daniell | Jul 2011 | A1 |
| 20130281671 | Peters et al. | Oct 2013 | A1 |
| 20150030575 | Daniell | Jan 2015 | A1 |
| 20150273082 | Nathwani et al. | Oct 2015 | A1 |
| 20150361158 | Tan et al. | Dec 2015 | A1 |
| 20160289277 | Herzog et al. | Oct 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| 2005185182 | Jul 2005 | JP |
| 2007053731 | May 2007 | WO |
| Entry |
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| Number | Date | Country | |
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| 20190194679 A1 | Jun 2019 | US |
| Number | Date | Country | |
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| 62310788 | Mar 2016 | US |
