Patent Application
20230151385
The present invention relates generally to vaccines and more specifically to non-naturally occurring, selectable marker (SM) proteins and methods of use thereof.
The creation of transgenic mammalian cell lines was pioneered almost four decades ago by Berg and colleagues (1-4). In general, this process involves transfecting (or transducing) cells with a recombinant DNA vector that carries the gene of interest and a selectable marker gene and then selecting for transgene-expressing cells using an appropriate antibiotic (3,4). This approach has been used thousands of times to create useful transgenic cell lines and is still in use today. However, most of the antibiotic-resistant clones arising from such experiments express low or undetectable levels of the linked transgene, requiring the isolation, expansion, and screening of dozens of independent, antibiotic-resistant, single-cell clones to find one that displays the desired level of transgene expression (5).
Many researchers have devoted significant effort towards improving the outcomes of mammalian cell transgenesis experiments. Much of this effort has been directed at improved vector design, resulting in the identification of many cis-acting features that affect transgene expression, including transcriptional regulatory regions (6-10), mRNA polyadenylation sites(11), introns (12), and mRNA export and/or translation signals (e.g. the Woodchuck Hepatitis Virus (WHP) posttranscriptional regulatory element (WPRE) (13)), and inhibitors of gene silencing(10,14). Other studies have revealed that gene silencing can be induced where bacterial and viral sequences in the transgenesis vectors can induce transgene silencing(15,16), effects that can be minimized by gene delivery via DNA mini-circles(17) or DNA transposons (e.g. Sleeping Beauty(18,19) or PiggyBac(20,21)). Furthermore, transgene delivery via replicating episomes (e.g. plasmids carrying Epstein-Barr virus (EBV) origin of replication (OriP) and the EBV nuclear antigen 1 gene (EBNA1)) may boost transgene expression via elevated gene dosage effects (22). Researchers have also invented technologies that direct transgene integration into sites of the host cell chromosome that are compatible with high-level, stable transgene expression, such as recombinase-mediated cassette exchange(23), phage ϕC31-mediated DNA integration(24), and CRISPR/Cas9-mediated genome editing(25). Although these site-directed insertional strategies represent a significant technological advance, several are limited to specific, previously engineered recipient cell lines, and all require the isolation, expansion, and characterization of numerous single cell clones.
Given the importance of mammalian cell transgenesis to biomedical research, it is somewhat surprising that there is as yet no robust body of empirical studies comparing the relative effectiveness of the dominant selectable markers that are typically used in these experiments. Currently there are five dominant selectable markers in widespread use, the NeoR, BsdR, HygR, PuroR, and BleoR genes, which confer resistance to the drugs G418, blasticidin, hygromycin B, puromycin, and zeocin, respectively (3,26-29). However, there is no clear understanding of whether they all work equally well for mammalian cell transgenesis experiments, or if they have differential effects on transgene expression. As a result, the choice of selectable marker in many mammalian cell transgenesis experiments tends to be based on circumstance rather than evidence.
Consequently, there is a pressing need to develop new expression vectors with improved selectable markers that could have a significant effect on the expression of recombinant proteins in both cells and exosomes. The present inventors sought to understand how the choice of selectable marker and the process of antibiotic selection affects the expression of linked recombinant proteins. The present inventors performed the studies described below in HEK293 cell lines because they are commonly used for biochemical and cell biological studies and are an approved cell factory for producing biological materials & drugs (5,30). Furthermore, the present inventors performed many of these studies in the context of a recombinant exosomal cargo protein, CD81, as the present inventors have a longstanding interest in exosome biology and exosome engineering (31-44).
Exosomes are small, secreted vesicles of ˜30-150 nm in diameter that are released by all human cell types, contain discrete subsets of proteins, nucleic acids, and lipids, and can transmit signals and molecules to other cells in a pathway of intercellular vesicle traffic, and are of increasing use as potential therapeutics and drug delivery vehicles(42).
Vaccination is an effective way to provide prophylactic protection against infectious diseases, including, but not limited to, viral, bacterial, and/or parasitic diseases, such as influenza, AIDS, hepatitis virus infection, cholera, malaria, tuberculosis, and many other diseases. For example, influenza infections are the seventh leading cause of death in the United States with 200,000 hospitalizations and 40,000 deaths seen in the United States per year and cause about 3-5 million hospitalizations and about 300,000 to 500,000 deaths worldwide per year. Millions of people receive flu vaccines to protect them from seasonal flu each year. Vaccination also holds the best potential for preventing the spread of coronavirus disease 2019 (COVID-19) caused by the SARS coronavirus 2 (SARS-CoV-2).
A typical vaccine contains an agent that resembles the disease-causing agent, which could be a microorganism, such as bacteria, virus, fungi, parasites, or one or more toxins. The antigen or agent in the vaccine stimulates the body's immune system to recognize the agent as a foreign invader, generate cellular immune responses and antibody (humoral) immune responses against it, and thereby inhibit or destroy it, and develop a memory of it. The vaccine-induced memory enables the immune system to act quickly to protect the body from any of these agents that it later encounters. Vaccine production used in the art, e.g., antigen vaccine production, has several stages, including the generation of antigens, antigen purification, in some cases inactivation of infectious agents, and vaccine formulation. The first phase of this process is to generate the antigen through culturing viruses in cell lines, growing bacteria in bioreactors, producing recombinant proteins derived from viruses and bacteria in cell cultures, yeast or bacteria, or synthesize nucleic acids that express the antigens of interest (AOIs). The second phase of the process is to purify the source of antigen, and in the case of killed agent vaccines, to inactivate the virus, bacteria, or parasite. The third phase of the process is to create the actual vaccine formulation, which may include anything from simple dilution of the AOI in buffer, to the mixing of the AOI with adjuvants.
As demonstrated by the COVID-19 outbreak, vaccine development can be a costly, time consuming endeavor that is outpaced by fast-spreading infections. There is therefore a great need for the development of new vaccine production platform technologies that can be modularly adapted to any new infectious agent. This need is also apparent for older infectious agents that mutate so rapidly that new vaccines are needed every year, as is the case for flu. Furthermore, we need improved technologies that generate vaccines that more closely mimic the physicochemical state of the intact virus, which is often vesicular in nature.
Consequently, there is a pressing need to develop new vaccines as well as new approaches to combatting infectious diseases.
The present invention provides, in a first aspect, an engineered, or non-naturally occurring, selectable marker (SM) protein, wherein the SM protein comprises a destabilization domain (DD) appended to, or operatively linked to, a SM protein, thereby providing a DD-tagged SM protein, or a degron-tagged, or a non-naturally occurring, SM protein. In some embodiments, the engineered SM protein is for recombinant protein expression. In some embodiments, said SM protein is a SM protein for, or that functions in, mammalian cells, e.g., human cells. In some embodiments, said DD is appended to the N-terminus of said SM protein, the C-terminus of said SM protein, or both the N-terminus and the C-terminus of said SM protein. In some embodiments, said DD is appended to the N-terminus of said SM protein. In some embodiments, said SM protein is a dominant SM protein. In some embodiments, said do SM protein confers resistance to zeocin, puromycin, hygromycin, G418, and/or blasticidin. In some embodiments, said SM protein that confers resistance to zeocin is BleoR. In some embodiments, said SM protein that confers resistance to puromycin is PuroR. In some embodiments, said SM protein that confers resistance to hygromycin is HygR. In some embodiments, said SM protein that confers resistance to G418 is NeoR. In some embodiments, said SM protein that confers resistance to blasticidin is BsdR. In some embodiments, said DD is derived from the human estrogen receptor (ER50), thereby providing a SM protein operably connected to the ER50(DD). In some embodiments, said SM protein operably connected to the ER50(DD) is BleoR operably connected to the ER50(DD), i.e., ER50BleoR. In some embodiments, said SM protein operably connected to the ER50(DD) is PuroR operatively connected to the ER50(DD), i.e., ER50PuroR. In some embodiments, said SM protein operably connected to the ER50(DD) is HygR operatively connected to the ER50(DD), i.e., ER50HygR. In some embodiments, said SM protein operably connected to the ER50(DD) is NeoR operatively connected to the ER50(DD), i.e., ER50NeoR. In some embodiments, said SM protein operably connected to the ER50(DD) is BsdR operatively connected to the ER50(DD), i.e., ER50BsdR. In some embodiments, said DD is derived from the human estrogen receptor (ER50), thereby providing an ER50(DD)-tagged SM protein. In some embodiments, said ER50(DD)-tagged SM protein is ER50(DD)-tagged BleoR (ER50BleoR), ER50(DD)-tagged PuroR (ER50PuroR), ER50(DD)-tagged HygR (ER50HygR), ER50(DD)-tagged NeoR (ER50NeoR), or ER50(DD)-tagged BsdR (ER50BsdR). In some embodiments, said DD is derived from the Escherichia coli dihydrofolate reductase (ecDHFR), thereby providing an SM protein operatively connected to the ecDHFR(DD). In some embodiments, said SM protein operably connected to the ecDHFR(DD) is BleoR operatively linked to the ecDHFR(DD), i.e., ecDHFRBleoR. In some embodiments, said SM protein operably connected to the ecDHFR(DD) is PuroR operatively linked to the ecDHFR(DD), i.e., ecDHFRPuroR. In some embodiments, said SM protein operably connected to the ecDHFR(DD) is HygR operatively linked to the ecDHFR(DD), i.e., ecDHFRHygR. In some embodiments, said SM protein operably connected to the ecDHFR(DD) is NeoR operatively linked to the ecDHFR(DD), i.e., ecDHFRNeoR. In some embodiments, said SM protein operably connected to the ecDHFR(DD) is BsdR operatively linked to the ecDHFR(DD), i.e., ecDHFRBsdR. In some embodiments, said DD is derived from the Escherichia coli dihydrofolate reductase (ecDHFR), thereby providing an ecDHFR(DD)-tagged SM protein. In some embodiments, said ecDHFR(DD)-tagged SM protein is ecDHFR(DD)-tagged BleoR (ecDHFRBleoR), ecDHFR(DD)-tagged PuroR (ecDHFRPuroR), ecDHFR(DD)-tagged HygR (ecDHFRHygR), ecDHFR(DD)-tagged NeoR (ecDHFRNeoR), or ecDHFR(DD)-tagged BsdR (ecDHFRBsdR). In some embodiments, the engineered SM protein further comprises an altered amino acid sequence resulting from a frameshift mutation within a nucleotide sequence that encodes the last about 10, 20, 30, 40, or 50 amino acids at the 3′ end of the DD-tagged SM.
The present invention provides a nucleic acid, the nucleotide sequence of which encodes the engineered SM protein according to the present invention. In some embodiments, said nucleic acid is an isolated nucleic acid.
The present invention provides an expression vector comprising a nucleic acid, the nucleotide sequence of which encodes a selectable marker (SM) protein and an operably linked recombinant protein of interest (POI), wherein the nucleic acid is operably linked to an expression control sequence. In some embodiments, the SM protein is unstable and/or degraded. In some embodiments, the SM protein selects for high-level expression of the linked recombinant POI. In some embodiments, said expression control sequence is a promoter. In some embodiments, said promoter is a cytomegalovirus (CMV) promoter. In some embodiments, said nucleic acid comprises an open reading frame (ORF), e.g., a bicistronic ORF, that encodes (a) the POI, followed by (b) a self-cleaving peptide which can induce ribosomal skipping during translation, and (c) the SM protein. In some embodiments, said self-cleaving peptide which can induce ribosomal skipping during translation is an about 18-22 amino acid-long peptide. In some embodiments, said about 18-22 amino acid-long peptide which can induce ribosomal skipping during translation is a 2a self-cleaving peptide. In some embodiments, said 2a self-cleaving peptide is p2a, e2a, f2a or t2a. In some embodiments, said 2a self-cleaving peptide is a viral 2a peptide. In some embodiments, said SM protein is PuroR2, having the amino acid sequence according to SEQ ID NO:1 (Table A), or a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:1. In some embodiments, said SM protein is HygR2, having the amino acid sequence according to SEQ ID NO:2 (Table A), or a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:2. In some embodiments, said SM protein is NeoR2, having the amino acid sequence according to SEQ ID NO:3 (Table A), or a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:3. In some embodiments, said SM protein is BsdR2, having the amino acid sequence according to SEQ ID NO:4 (Table A), or a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:4. In some embodiments, said SM protein is BsdR5, having the amino acid sequence according to SEQ ID NO:5 (Table A), or a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:5.
In some embodiments of the invention, said SM protein is the engineered SM protein according to the present invention. In some embodiments, said POI is a transcriptional activator (TA). In some embodiments, said transcriptional activator is a reverse tetracycline transcriptional activator (rtTA). In some embodiments, said rtTA is rtTAv16 or rtTAv16/G72P. In some embodiments, said POI is an antigen of a pathogen. In some embodiments, said antigen of a pathogen is SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, or envelope (E) protein. In some embodiments, said antigen of a pathogen is SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In one aspect, the expression vector POI comprises a plasma membrane anchor and/or an oligomerization domain.
The present invention provides, in another aspect, PuroR2 gene which encodes a GNAT family N-acetyltransferase protein from Streptomyces rimosus (WP_125058166.1), as well genes, codon optimized or otherwise, that encode a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:1.
The present invention provides, in another aspect, HygR2 gene which encodes an aminoglycoside-O-phosphotransferase protein from Ochrobactrum cytisi (WP_071631649.1), as well genes, codon optimized or otherwise, that encode a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:2.
The present invention provides, in another aspect, NeoR2 gene which encodes an aminoglycoside-O-phosphotransferase protein from Nitrosomonas oligotropha (WP_107804178.1), as well genes, codon optimized or otherwise, that encode a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:3.
The present invention provides, in another aspect, a selectable marker (SM) protein comprising C-terminal 17 amino acids of a 2a self-cleaving peptide, wherein said C-terminal 17 amino acids of a 2a self-cleaving peptide is tagged to the C-terminus of the SM.
The present invention provides, in another aspect, a protein of interest (POI) comprising an N-terminal proline of a 2a self-cleaving peptide, wherein said N-terminal proline of a 2a self-cleaving peptide is tagged to the N-terminus of the POI.
The present invention provides, in another aspect, BsdR2 gene which encodes a GNAT family N-acetyltransferase from Pantoea ananatis (WP_024470972.1), as well genes, codon optimized or otherwise, that encode a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:4.
The present invention provides, in another aspect, BsdR5 gene which encodes a small molecules deaminase enzyme from Aspergillus udagawae (WP_024470972.1), or genes that encode a protein or a polypeptide sharing or having 60% or greater amino acid sequence identity with, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% amino acid sequence identity with, SEQ ID NO:5.
The present invention provides a cell comprising the expression vector according to the present invention.
The present invention provides a cultured cell line comprising the expression vector according to the present invention, wherein the cells in the cultured cell line are selected by selection, or culturing, in a selector-containing media. In some embodiments, said selector-containing media is an antibiotic-containing media. In some embodiments, said antibiotic-containing media contains zeocin, puromycin, hygromycin, G418, and/or blasticidin.
The present invention provides a method of making a cell line comprising culturing cells transfected with the expression vector according to the present invention with a selector for the selectable marker. In some embodiments, said selector is an antibiotic. In some embodiments, said antibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin. In one aspect the celltured in the presence of a selector for the selectable marker.
The present invention provides a method of making extracellular vesicles (EVs) comprising culturing the cell line according to the invention, wherein the cell line produces EVs comprising one or more of the POIs and isolating the EVs produced. In some embodiments, said EVs are exosomes or microvesicles.
The present invention provides a cultured cell line comprising cells comprising the expression vector according to the invention, wherein the cells in the cell line have less cell-to-cell variance in POI production compared to cells which are not transfected with the expression vector according to the invention. In some embodiments, the parental cell line is 293F (Ftet cell line). In some embodiments, the cultured cell line according to the present aspect of the present invention is a mixed-clone (Ftet1) cell line of the Ftet cell line comprising a plurality of the Ftet cells which are resistant to a selector. In some embodiments, said selector is an antibiotic. In some embodiments, said antibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin. In some embodiments, the cultured cell line according to the present aspect of the present invention is a single cell clone (Ftet2) cell line of the Ftet cell line comprising a single cell clone (SCC) exhibiting the highest relative expression of the transcriptional activator.
The present invention provides an expression vector comprising: (a) a nucleic acid the nucleotide sequence of which encodes a transposon comprised of inverted terminal repeats (ITR-L and ITR-R elements) that define the left and right ends of the transposon; (b) one or more genes of interest (GOIs) encoding one or more proteins of interest (POIs) inserted between the ITR-L and ITR-R elements, wherein the GOIs are operably linked to expression control sequences; and (c) a nucleic acid the nucleotide sequence of which encodes a transposase enzyme, wherein the nucleic acid is located outside the transposon, and wherein the nucleic acid is operably linked to an expression control sequence. In some embodiments, the expression vector according to the aspect of the present invention further comprises (d) a nucleic acid, the nucleotide sequence of which encodes a selectable marker (SM) protein, wherein the nucleic acid is operably linked to an expression control sequence. In some embodiments, said SM protein selects for high-level expression of the GOIs. In some embodiments, said SM protein is an unstable and/or degraded SM protein. In some embodiments, the nucleic acid of element (d) is inserted between the ITR-L and ITR-R elements. In some embodiments, said expression control sequences operably linked to the GOIs include a plurality of binding sites for binding to a transcriptional activator, whereby the number of the binding sites can be altered to modulate the POI stoichiometry. In some embodiments, said transcriptional activator is reverse tetracycline transcriptional activator (rtTA). In some embodiments, said rtTA is rtTAv16 or rtTAv16/G72P. In some embodiments, said expression control sequence is a promoter. In some embodiments, said promoter of element (d) is an EFlalpha (or its short, intron-less form, EFS) promoter. In some embodiments, said expression control sequences of element (b) are inducible promoters, thereby providing an expression vector for inducible expression of GOIs (inducible expression vector). In some embodiments, said inducible promoters are induced by tetracycline or doxycycline. In some embodiments, said inducible promoters are TRE3G promoters. In some embodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E) proteins. In some embodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In some embodiments, said S protein is the Wuhan-1 strain S protein; a furin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein; or the Wuhan-1 strain S protein with an amino acid change of D614G. In some embodiments, said transposase enzyme is a Sleeping Beauty (SB) transposase enzyme. In some embodiments, said nucleic acid the nucleotide sequence of which encodes a transposase enzyme comprises a gene configured to express an optimized version of the SB transposase enzyme. In some embodiments, said gene configured to express an optimized version of the SB transposase enzyme is RSV-SB100x-pAn. In some embodiments, said SM protein is PuroR2. In some embodiments, said SM protein is the SM protein according to the present invention.
The present invention provides the cultured cell line according to the present invention, wherein the cultured cell line is further transfected with the expression vector according to the ninth aspect of the present invention. In some embodiments, the cultured cell line is further transfected with two or more of the expression vectors of the present invention, wherein each of said two or more of the expression vectors according to the ninth aspect of the present invention has a separate selectable marker.
The present invention provides a method of making a cell line comprising transfecting the cultured cell line according to the present invention with the expression vector according to the present invention.
The present invention provides a method of making a cell line comprising transfecting the cultured cell line according to according to the present invention with two or more of the expression vectors according to the present invention, wherein each of said two or more of the expression vectors according to the present invention has a separate selectable marker.
The present invention provides an extracellular vesicle-based therapeutic or prophylactic composition comprising a plurality of extracellular vesicles (EVs) derived from the cultured cell line according to the present invention, wherein said GOIs encode one or more therapeutic or prophylactic proteins of interest (POIs). In some embodiments, said one or more therapeutic or prophylactic POIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E) proteins. In some embodiments, said one or more therapeutic or prophylactic POIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In some embodiments, the EV-based therapeutic or prophylactic composition according to the present aspect of the present invention further comprises a physiologically acceptable excipient and/or adjuvant.
The present invention provides an expression vector comprising: (a) a nucleic acid the nucleotide sequence of which encodes a first transposon comprised of inverted terminal repeats (ITR-L and ITR-R elements) that define the left and right ends of the first transposon; (b) one or more genes of interest (GOIs) encoding one or more proteins of interest (POIs) inserted between the ITR-L and ITR-R elements of the first transposon, wherein the GOIs are operably linked to expression control sequences; (c) a nucleic acid the nucleotide sequence of which encodes a second transposon comprised of ITR-L and ITR-R elements that define the left and right ends of the second transposon; (d) a nucleic acid the nucleotide sequence of which encodes a transcriptional activator, wherein the nucleic acid is inserted between the ITR-L and ITR-R elements of the second transposon; and (e) a nucleic acid the nucleotide sequence of which encodes a transposase enzyme, wherein the nucleic acid is located outside the first and second transposons, and wherein the nucleic acid is operably linked to an expression control sequence. In some embodiments, the expression vector according to the present aspect of the present invention further comprises (f) a nucleic acid the nucleotide sequence of which encodes a selectable marker (SM) protein that selects for high-level expression of the GOIs. In some embodiments, said SM protein is an unstable and/or degraded SM protein. In some embodiments, the nucleic acid of element (f) is inserted between the ITR-L and ITR-R elements of the first transposon. In some embodiments, said expression control sequences operably linked to the GOIs include a plurality of binding sites for binding to the transcriptional activator, whereby the number of the binding sites can be altered to modulate the POI stoichiometry. In some embodiments, said transcriptional activator is reverse tetracycline transcriptional activator (rtTA). In some embodiments, said rtTA is rtTAv16 or rtTAv16/G72P. In some embodiments, the expression control sequences of element (b) are inducible promoters, thereby providing an expression vector for inducible expression of GOIs (inducible expression vector). In some embodiments, said inducible promoters are induced by tetracycline or doxycycline. In some embodiments, said inducible promoters are TRE3G promoters. In some embodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E) proteins. In some embodiments, said GOIs encode SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In some embodiments, said S protein is the Wuhan-1 strain S protein; a furin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein; or the Wuhan-1 strain S protein with an amino acid change of D614G. In some embodiments, said transposase enzyme is a Sleeping Beauty (SB) transposase enzyme. In some embodiments, said nucleic acid the nucleotide sequence of which encodes a transposase enzyme comprises a gene configured to express an optimized version of the SB transposase enzyme. In some embodiments, said gene configured to express an optimized version of the SB transposase enzyme is RSV-SB100x-pAn. In some embodiments, said SM protein is PuroR2. In some embodiments, said SM protein is the SM protein according to the present invention. In one aspect, wherein the POI comprises a plasma membrane anchor and/or an oligomerization domain.
The present invention provides a cell comprising the expression vector according to the present invention.
The present invention provides a cultured cell line comprising the expression vector according to the present invention, wherein the cells in the cultured cell line are selected by culturing in a selector-containing media. In some embodiments, said selector-containing media is an antibiotic-containing media. In some embodiments, said antibiotic-containing media contains zeocin, puromycin, hygromycin, G418, and/or blasticidin.
The present invention provides a method of making a cell line comprising culturing cells transfected with the expression vector according to an aspect of the present invention with a selector for the selectable marker. In some embodiments, said selector is an antibiotic. In some embodiments, said antibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin.
The present invention provides a method of making extracellular vesicles (EVs) comprising culturing a cell line according to the present invention, wherein the cell line produces EVs comprising one or more of the POIs and isolating the EVs produced. In some embodiments, said EVs are exosomes or microvesicles.
The present invention provides a cultured cell line comprising cells comprising the expression vector according to the present invention, wherein the cells in the cell line have less cell-to-cell variance in POI production compared to cells which are not transfected with the expression vector according to the present invention. In some embodiments, the parental cell line is 293F (Ftet cell line). In some embodiments, the cultured cell line according to the present aspect of the present invention is a mixed-clone (Ftet1) cell line of the Ftet cell line comprising a plurality of the Ftet cells which are resistant to a selector. In some embodiments, said selector is an antibiotic. In some embodiments, said antibiotic is zeocin, puromycin, hygromycin, G418, or blasticidin. In some embodiments, the cultured cell line according to the present aspect of the present invention is a single cell clone (Ftet2) cell line of the Ftet cell line comprising a single cell clone (SCC) exhibiting the highest relative expression of the transcriptional activator. culturing the cells in the presence of a selector for the selectable marker.
The present invention provides a pharmaceutical composition comprising a plurality of extracellular vesicles (EVs) derived from the cultured cell line according to the present invention, wherein said POI is a therapeutic or prophylactic POI. In some embodiments, said POI is a therapeutic or prophylactic POI. In some embodiments, said prophylactic POI is an antigen of interest (AOI). In some embodiments, one or more antigens of interest (AOIs) are displayed on the surface of the EVs. In some embodiments, said AOIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E) proteins. In some embodiments, said AOIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In some embodiments, said S protein is the Wuhan-1 strain S protein; a furin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein; or the Wuhan-1 strain S protein with an amino acid change of D614G. In some embodiments, the pharmaceutical composition according to the present aspect of the present invention further comprises a physiologically acceptable excipient and/or adjuvant. In some embodiments, said EVs are exosomes or microvesicles.
The present invention provides a method of preventing a viral infection comprising administering to a subject susceptible to a viral infection an immunogenically effective amount of the pharmaceutical composition according to an aspect of the present invention.
The present invention provides an extracellular vesicle (EV)-based antigen display composition comprising: an EV displaying one or more antigens of interest (AOIs). In some embodiments, the EV-based antigen display composition according to the present aspect of the present invention is an EV-based antigen display vaccine. In some embodiments, the EV-based antigen display composition according to the present aspect of the present invention is an immune response stimulating composition. In some embodiments, said AOIs are displayed on the surface of the EVs. In some embodiments, said AOIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and/or envelope (E) proteins. In some embodiments, said AOIs are SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In some embodiments, said S protein is the Wuhan-1 strain S protein; a furin-blocked, trimer-stabilized form of the Wuhan-1 strain S protein; or the Wuhan-1 strain S protein with an amino acid change of D614G. In some embodiments, said EVs are exosomes or microvesicles. In one aspect, the EV-based antigen display composition is an immune response stimulating composition. In one aspect, the EV-based antigen display composition is a pharmaceutical composition including a physiologically acceptable excipient and/or adjuvant. In one aspect, the EV-based antigen display composition is an immune response stimulating composition.
In one aspect, the invention provides a composition comprising extracellular vesicles (EVs) containing, as cargo (e.g., internally or on their surface) a protein of interest. For example, the protein of interest comprises SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In another aspect, the protein of interest comprises an EV-trafficking element. Further, a composition of the invention can be formulated for administration to an animal (e.g., a human) subject via an oral route, a sublingual route, a buccal route, a rectal route, a topical route, a transdermal route, injection, inhalation or a pulmonary route.
The present invention provides a pharmaceutical composition comprising the EV-based antigen display composition according to the present invention, and further comprising a physiologically acceptable excipient and/or adjuvant.
The present invention provides a method of preventing a viral infection comprising administering to a subject susceptible to a viral infection an immunogenically effective amount of the EV-based antigen display composition according to the present invention, or the pharmaceutical composition according to the invention.
The present invention provides a composition comprising a population of extracellular vesicles comprising a protein of interest (POI), wherein the EVs in the population have higher POI expression compared with an EV population made from cells not transfected with a selectable marker and selected for the selected marker, as disclosed herein, or have a lower EV-to-EV variation (e.g., at least 25%, 50%, 75%, 80% or 90% less variation) in POI expression compared with an EV population made from cells not transfected with a selectable marker and selected from the selected marker, as disclosed herein.
In one embodiment, the invention provides a composition including extracellular vesicles (EVs) containing, as cargo (e.g., internally or on their surface) a protein of interest (POI). For example, a POI may include SARS-CoV-2 spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, envelope (E) protein, orf3a-encoded protein, and/or orf7a-encoded protein. In one aspect, the protein of interest comprises an EV-trafficking element. In another aspect, the composition is formulated for administration to an animal (e.g., a human) subject via an oral route, a sublingual route, a buccal route, a rectal route, a topical route, a transdermal route, injection, inhalation or a pulmonary route.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
The terms “treat”, “therapeutic”, “prophylactic” and “prevent” are not intended to be absolute terms. Treatment, prevention and prophylaxis can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. Treatment, prevention, and prophylaxis can be complete or partial. The term “prophylactic” means not only “prevent”, but also minimize illness and disease. For example, a “prophylactic” agent can be administered to subject to prevent infection, or to minimize the extent of illness and disease caused by such infection. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects, the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.
A treatment can be considered “effective,” as used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 2%, 3%, 4%, 5%, 10%, or more, following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (e.g., progression of the disease is halted). Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. One skilled in the art can monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters.
The term “effective amount” as used herein refers to the amount of a composition or an agent needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of therapeutic composition to provide the desired effect. The term “therapeutically effective amount” refers to an amount of a composition or therapeutic agent that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, can include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of therapeutic effect at least any of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least any of a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The therapeutically effective amount may be administered in one or more doses of the therapeutic agent. The therapeutically effective amount may be administered in a single administration, or over a period of time in a plurality of doses.
“Administering” as used herein can include any suitable routes of administering a therapeutic agent or composition as disclosed herein. Suitable routes of administration include, without limitation, oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection or topical administration. Administration can be local or systemic.
As used herein, the term “pharmaceutically acceptable” refers to a carrier that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. For the present invention, the dose can refer to the concentration of the extracellular vesicles or associated components, e.g., the amount of therapeutic agent or dosage of radiolabel. The dose will vary depending on a number of factors, including frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; the route of administration; and the imaging modality of the detectable moiety (if present). One of skill in the art will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical, and depends on the route of administration. For example, a dosage form can be in a liquid, e.g., a saline solution for injection.
“Subject,” “patient,” “individual” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision. A patient can be an individual that is seeking treatment, monitoring, adjustment or modification of an existing therapeutic regimen, etc.
As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of” between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1, 2 or 3” means “at least 1, at least 2 or at least 3”. The phrase “at least one” includes “a plurality”.
Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-91 1910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
The term “selectable marker” (SM) refers to a gene introduced into a cell that confers a trait suitable for artificial selection. Examples of selectable markers includes antibiotic resistance markers, such as BleoR (zeocin resistance), PuroR (puromycin resistance), HygR (hygromycin resistance), NeoR (G418 resistance), BsdR (blasticidin resistance), AmpR (ampicillin resistance), TetR (tetracycline resistance), and KanR (kanamycin resistance).
The term “dominant selectable marker” refers to a protein that is encoded by a conditionally dominant gene introduced into a cell that confers an ability to grow in the presence of applied selective agents that are normally toxic to cells or inhibitory to cell growth, such as antibiotics.
The term “destabilization domain” (DD) refers to a protein, polypeptide or amino acid sequence that modulates the stability of a protein when operably connected to, linked to, or fused to (e.g., as a fusion component of), the protein. For example, the term “destabilization domain” (DD), refers to a protein domain that is unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. Destabilization domains (DDs) can be fused or linked to a target protein and can convey its destabilizing property to the protein of interest, causing protein degradation. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell. However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and some level of protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Such a destabilization domain may or may not require the interaction of another protein for modulating stability of the protein. Non-limiting examples of DDs include structurally unstable protein domains derived from Escherichia coli dihydrofolate reductase (DHFR), as described in Iwamoto M, Björklund T, Lundberg C, Kirik D, Wandless T J. Chem Biol. 2010; 17:981-988, and the human estrogen receptor (ER50), as described in Miyazaki Y, Imoto H, Chen L C, Wandless T J. J Am Chem Soc. 2012; 134:3942-3945, as well as Maji B, Moore C L, Zetsche B, Volz S E, Zhang F, Shoulders M D, Choudhary A. Multidimensional chemical control of CRISPR-Cas9. Nat Chem Biol. 2017 January; 13(1):9-11, the contents of which are each incorporated herein by reference in their entirety. The term “extracellular vesicle” (EV) refers to lipid bilayer-delimited particles that are naturally released from cells. EVs range in diameter from around 20-30 nanometers to about 10 microns or more. EVs can comprise proteins, nucleic acids, lipids and metabolites from the cells that produced them. EVs include exosomes (about 50 to about 100 nm), microvesicles (about 100 to about 300 nm), ectosomes (about 50 to about 1000 nm), apoptotic bodies (about 50 to about 5000 nm) and lipid-protein aggregates of the same dimensions. In another example, the destabilization domain can comprise KEN, Cyclin A, UFD domain/substrate, ubiquitin, PEST sequences, destruction boxes and hydrophobic stretches of amino acids. Exemplary destabilization domains include ubiquitin and homologs thereof, particularly those comprising mutations that prevent, or significantly reduce, the cleavage of ubiquitin multimers by a-NH-ubiquitin protein endoproteases.
The term “virus-like particle” or “VLP” is meant to refer to any supramolecular nanoparticle (ranging from 20 to 100 nm) that comprises envelope and/or capsid viral proteins, but that is non-infectious because it does not contain viral genetic material. VLPs are useful as part of vaccine composition, as they contain repetitive, high density displays of viral surface proteins presenting conformational viral epitopes that can elicit T cell and B cell immune responses. Since VLPs cannot replicate, they provide a safer alternative to attenuated viruses. EVs as described herein, which include molecules that resemble viruses but that are not infectious and non-replicative can be used as VLPs.
Extracellular vesicles and virus-like particles can be referred to herein as “delivery vehicles.” An extracellular vesicle can carry a cargo, which can be a protein of interest (POI) or a nucleic acid of interest (NAOI). The cargo molecule can be present within the lumen of the delivery vehicle or on its surface. The protein of interest can be a protein that is naturally produced by a cell that generates a delivery vehicle, or it can be a recombinant protein, including a non-naturally occurring protein, such as a fusion protein. The POI can be a viral protein, e.g., capable of eliciting an immune response. Nucleic acids include, without limitation, DNA and RNA. RNA can be mRNA. When delivered to a target cell, mRNA may be expressed as protein and presented on the cell surface to elicit an immune response. Nucleic acids are typically incorporated into EVs by contacting the EVs and the nucleic acid in the presence of a chemical lipofection reagent. The chemical lipofection reagent can be a polycationic lipid. In some embodiments, the chemical lipofection reagent is an mRNA lipofection reagent, or an mRNA transfection reagent, e.g., Lipofectamine® MessengerMAX™, Lipofectamine® 2000, Lipofectamine® 3000.
The nucleotide and amino acid sequences of the SARS-CoV-2 Wuhan-1 strain spike (S) protein, and the Wuhan-1 strain S protein with an amino acid change of D614G are well known in the art, and are described in, e.g., Plante, J. A., Liu, Y., Liu, J. et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature (2020), the contents of which are each incorporated herein by reference in their entirety.
The term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, anti-sense DNA strands, shRNA, ribozymes, nucleic acids conjugated and oligonucleotides. According to the invention, a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid might be employed for introduction into, e.g., transfection of, cells, e.g., in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation. Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, N.Y. 2,028 pages (2012).
A SARS-CoV-2 virion is approximately 50-200 nanometers in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the complete viral envelope, which are the proteins of interest in regard to the present invention. The spike protein, S, which has been imaged at the atomic level using cryogenic electron microscopy, is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell. As used herein, the phrase SARS-CoV-2 structural “protein S, N, M, and/or E” refers to the spike (S), nucleocapsid (N), membrane (M), and/or envelope (E) proteins, respectively, which are encoded by the nucleic acid sequences of the invention, or by a codon-optimized oligonucleotide sequence, encoding each protein individually, or any combination of 2 or 3 proteins, or a combination of all 4 proteins. When two or more nucleic acid sequences are included in a single vector or construct, they are in operable linkage such that the each of the 2, 3, or 4 SARS-CoV-2 structural proteins are properly encoded and expressed. Nucleic acid sequences encoding additional SARS-CoV-2 proteins, such as orfa or orfa/b polypeptides are also included in the nucleic acid sequences of the present invention. Such nucleic acid sequences may be incorporated in a vector as described herein to provide a variation of these vectors. Cells transfected with a vector as described herein, may be transfected with a vector including a nucleic acid sequence encoding an additional SARS-CoV-2 protein.
The term “vector”, “expression vector”, or “plasmid DNA” is used herein to refer to a recombinant nucleic acid construct that is manipulated by human intervention. A recombinant nucleic acid construct can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature. For instance, the two or more nucleotide sequences can be operatively linked, such as one or more genes encoding one or more proteins of interest, one or more protein tags, functional domains and the like. For example, the proteins of interest according to the invention can include SARS-CoV-2 structural protein S, N, M, and/or E. The expression vector of the invention can include regulatory elements controlling transcription generally derived from mammalian, microbial, viral or insect genes, such as an origin of replication to confer the vector the ability to replicate in a host, and a selection gene encoding, e.g., a selectable marker (SM) protein, to facilitate recognition of transformants may additionally be incorporated. Those of skill in the art can select a suitable regulatory region to be included in such a vector depending on the host cell used to express the gene(s). For example, the expression vector can comprise one or more promoters, operably linked to the nucleic acid of interest, or a gene of interest (GOI), capable of facilitating transcription of genes in operable linkage with the promoter. Several types of promoters are well known in the art and suitable for use with the present invention. The promoter can be constitutive or inducible. ADDitional regulatory elements that may be useful in vectors include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, introns, and the like. Such elements may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such elements can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, may sometimes be obtained without such additional elements. Vectors also can include other elements. For example, a vector can include a nucleic acid that encodes a signal peptide such that the encoded polypeptide is directed to a particular cellular location (e.g., a signal secretion sequence to cause the protein to be secreted by the cell) or a nucleic acid that encodes a selectable marker. Non-limiting examples of selectable markers include doxycycline, puromycin, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Non-limiting examples of vectors suitable for use for the expression of high levels of recombinant proteins of interest include those selected from baculovirus, phage, plasmid, phagemid, cosmid, fosmid, bacterial artificial chromosome, viral DNA, Pl-based artificial chromosome, yeast plasmid, transposon, and yeast artificial chromosome. For example, the viral DNA vector can be selected from vaccinia, adenovirus, foul pox virus, pseudorabies and a derivative of SV40. Non-limiting examples of suitable bacterial vectors include pQE70™, pQE60™, pQE-9™, pBLUESCRIPT™ SK, pBLUESCRIPT™ KS, pTRC99a™, pKK223-3™, pDR540™, PAC™ and pRIT2T™. Non-limiting examples of suitable eukaryotic vectors include pWLNEO™, pXTI™, pSG5™, pSVK3™, pBPV™, pMSG™, and pSVLSV40™. Non-limiting examples of suitable eukaryotic vectors include pWLNEO™, pXTI™, pSG5™, pSVK3™, pBPV™, pMSG™, and pSVLSV40™. One type of vector is a genomic integrated vector which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, e.g., a nucleic acid capable of extra-chromosomal replication. Viral vectors include adenovirus, adeno-associated virus (AAV), retroviruses, lentiviruses, vaccinia virus, measles viruses, herpes viruses, and bovine papilloma virus vectors (see, Kay et al., Proc. Natl. Acad. Sci. USA 94:12744-12746 (1997) for a review of viral and non-viral vectors). Viral vectors can be modified so the native tropism and pathogenicity of the virus are altered or removed. The genome of a virus also can be modified to increase its infectivity and to accommodate packaging of the nucleic acid encoding the polypeptide of interest.
The term “derived from” as in “A is derived from B” means that A is obtained from B in such a manner that A is not identical to B. For instance, if a destabilization domain (DD) is “derived from” the Escherichia coli dihydrofolate reductase (ecDHFR), that means the degradation domain of the ecDHFR is obtained from ecDHFR, thereby providing ecDHFR(DD). Accordingly, the term “ecDHFRBsdR” refers to BsdR which has been derivatized by appending the degradation domain of the ecDHFR to the N-terminus of BsdR. The term “polycistronic” (e.g., “bicistronic”) refers to a nucleic acid molecule, e.g., mRNA, which, upon translation, produces a plurality of polypeptides. A plurality of polypeptides can be produced by, for example, inclusion of a plurality of open reading frames, or a single reading frame comprising a self-cleaving peptide, such as viral 2A peptide.
The term “self-cleaving peptide” refers to a peptide that mediates ribosome-skipping events during translation, producing independent polypeptides from a single message. Examples of self-cleaving peptides include peptides of the 2A peptide family, including: T2A—EGRGSLL TCGDVEENPGP (thosea asigna virus 2A); P2A—ATNFSLLKQAGDVEENPGP (porcine teschovirus-1 2A); E2A—QCTNYALLKLAGDVESNPGP (equine rhinitis A virus) and F2A—VKQTLNFDLLKLAGDVESNPGP (foot-and-mouth disease virus 18). Adding the optional linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.
The term “transgene” refers to a gene in a cell or organism that is not native to the at cell or organism, typically incorporated naturally, or by any of a number of genetic engineering techniques.
The term “open reading frame” (ORF) refers to a nucleotide sequence, typically positioned between a start codon and a stop codon, that has the ability to be translated into a polypeptide.
The term “expression control sequence” refers to a nucleotide sequence that regulates transcription and/or translation of a nucleotide sequence operatively linked thereto. Expression control sequences include, but are not limited to, promoters, enhancers, repressors (transcription regulatory sequences) and ribosome binding sites (translation regulatory sequences).
As used herein, a nucleotide sequence is “operably linked” with an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.
The term “clone” refers to a group of identical cells that share a common ancestry, e.g., they are derived from the same cell.
A variety of host cells are known in the art and suitable for proteins expression and extracellular vesicles production. Non-limiting examples of typical cell used for transfection include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell. For example, human embryonic kidney 293 (HEK293), E. coli, Bacillus, Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2, Spodoptera SJ9, CHO, COS (e.g. COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, or 293F.
A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Non-limiting examples of typical non-viral mediated techniques includeelectroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include, but are not limited to, proprietary transfection reagents such as LIPOFECTAMINE™, DOJINDO HILYMAX™, FUGENE™, JETPEI™, EFFECTENE™ and DREAMFECT™.
A pathogen, which can be a bacteria, virus, or any other microorganism that can cause a disease in a subject, can elicit an immune response (i.e., an integrated bodily response to a pathogen antigen, which can include a cellular immune response and/or a humoral immune response) in the subject. For example, upon contact and/or exposure to a pathogen, a subject may respond with an humoral immune response, characterized by the production of antibody, specifically directed against one or more pathogen antigens.
As used herein the term “antibody” refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen. Antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. The light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 6, c, y, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The antibody may have one or more effector functions which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region or any other modified Fc region) of an antibody. Non-limiting examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor (BCR); and cross-presentation of antigens by antigen presenting cells or dendritic cells).
The term “neutralizing antibody” (Nab) refers an antibody that defends a cell from a pathogen or infectious particle by neutralizing any effect it has biologically. Neutralization renders the particle no longer infectious or pathogenic. Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria and microbial toxin. By binding specifically to surface antigen on an infectious particle, neutralizing antibodies prevent the particle from interacting with its host cells it might infect and destroy. Immunity due to neutralizing antibodies is also known as sterilizing immunity, as the immune system eliminates the infectious particle before any infection took place.
The term “antigen” refers to any substance that will elicit an immune response. For instance, an antigen relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells). As used herein, the term “antigen” comprises any molecule which comprises at least one epitope. For instance, an antigen is a molecule which, optionally after processing, induces an immune reaction. For instance, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction may be a cellular immune reaction. For instance, the antigen may be presented by a cell, which results in an immune reaction against the antigen. For example, an antigen is a product which corresponds to or is derived from a naturally occurring antigen. Such antigens include, but are not limited to, SARS-CoV-2 structural proteins S, N, M, and E, and any variants or mutants thereof.
The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to any chain of at least two amino acids, linked by a covalent chemical bound. As used herein a peptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof. A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
The term “pharmaceutical composition” refers to a formulation comprising an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient. The term “active ingredient” can interchangeably refer to an “effective ingredient,” and is meant to refer to any agent that is capable of inducing a sought-after effect upon administration. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, nor to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Examples of carrier include, but are not limited to, liposome, nanoparticles, ointment, micelles, microsphere, microparticle, cream, emulsion, and gel. Examples of excipient include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate and parabens. Examples of diluent include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil and dimethyl sulfoxide (DMSO).
The term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, e.g., a cellular immune response, which recognizes and attacks a pathogen or a diseased cell. The term “immune response” refers to an integrated bodily response to an antigen and refers to a cellular immune response and/or a humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.
A “cellular immune response” can include a cellular response directed to cells characterized by presentation of an antigen with class I or class II MEW, or a humoral response directed to the production of antibodies. The cellular response relates to cells called T cells or T-lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells.
The terms “immunoreactive cell” “immune cells” or “immune effector cells” relate to a cell which exerts effector functions during an immune reaction. An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen or an antigen peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, secrete antibodies, recognize cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.
The term “adjuvant” refers to a pharmacological or immunological agent that modifies the effect of other agents. An adjuvant may be added to the vaccine composition of the invention to boost the immune response to produce more antibodies and longer-lasting immunity, thus minimizing the dose of antigen needed. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells depending on the purpose of the vaccine. Immunologic adjuvants are added to vaccines to stimulate the immune system's response to the target antigen, but do not provide immunity themselves. Examples of adjuvants include, but are not limited to analgesic adjuvants; inorganic compounds such as alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide; mineral oil such as paraffin oil; bacterial products such as killed bacteria (Bordetella pertussis, Mycobacterium bovis, toxoids); nonbacterial organics such as squalene; delivery systems such as detergents (Quil A); plant saponins from Quillaja, soybean, or Polygala senega; cytokines such as IL-1, IL-2, IL-12; combination such as Freund's complete adjuvant, Freund's incomplete adjuvant; food-based oil such as Adjuvant 65, which is based on peanut oil.
The contents of exosomes depends, in part, on the character of the cells that produce them. Cells can be genetically modified to configure exosomes produced by them. Fang et al., (PLOS, June 2007 vol. 5:1267-1283) describe methods of engineering proteins to preferentially target them toward exosomes. It was observed that (1) addition of both monoclonal mouse IgG to CD43 and polyclonal anti-mouse IgG antibodies were sufficient to induce the sorting of CD43 to exosomes, (2) addition of a plasma membrane anchor was sufficient to target a protein to exosomes, (3) a synthetic cargo comprised of a plasma membrane anchor and two heterologous oligomerization domains (Acyl-LZ-DsRED) was sorted to exosomes, (4) highly oligomeric, plasma membrane-associated retroviral Gag proteins (from EIAV, HTLV-1, RSV, MLV, MPMV, and HERV-K) were all sorted to ELDs and exosomes, and (5) a pair of heterologous oligomerization domains was necessary and sufficient to target HIV Gag to ELDs and exosomes. Elements, such as these, that traffic proteins to EVs, are referred to as “EV-trafficking elements.” Accordingly, any protein of interest can be modified in this way to traffic the protein towards exosomes.
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.
For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. In order that the present invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
Plasmids were maintained in DH10B cells, grown in ampicillin-containing LB media, and purified from bacterial lysates using mini-prep and midi-prep plasmid isolation kits (Promega). Automated Sanger DNA sequencing was performed using custom primers and dye-linked dideoxynucleotides (Applied Biosystems). DNA sequence data were assembled, maintained and analyzed using SnapGene software. The 3×NLS-tdTomato, CD81mNG, BleoR, PuroR, HygR, BsdR, NeoR, and SB100X coding regions were codon-optimized for expression in human cells, synthesized in vitro, cloned into mammalian cell expression vectors, and sequence-confirmed prior to use. The ITR-left and ITR-right backbone of the Sleeping Beauty vectors(18,19) and the cis-acting elements of a third generation, replication-defective and self-inactivating lentiviral backbone(57) were also synthesized in vitro, cloned into minimal bacterial plasmids, and sequence-confirmed prior to use. The EBNA1 and OriP sequences(22) were assembled by a combination of in vitro gene synthesis and excision from pCEP4 (ThermoFisher), cloned into a minimal plasmid vector, with sequence confirmation of all newly synthesized segments of DNA. Transgenes and polylinkers were inserted into transgene delivery vectors using standard recombinant DNA cloning techniques.
To make replicating-defective, self-inactivating lentiviruses, 293T cells were transfected with a mixture of 4 plasmids: the lentiviral vector, a Gag-Pol expression vector, a Rev expression vector, and a VSV-G expression vector(57). The transfected cells were incubated for 3 days. The tissue culture supernatant was collected, spun at 5000×g for 15 minutes to remove cells and cell debris, and the resulting supernatant was passed through a 0.45 μm filter to generate an unconcentrated virus stock.
All cell lines were grown in a tissue culture incubator maintained at 5% CO2, 90-99% humidity, and 37° C. HEK293 and 293T cells were obtained from the American Type Culture Collection (ATCC) and grown in DMEM (high glucose; Gibco/BRL) supplemented with 10% fetal calf serum (FCS; ThermoFisher). COST cells were also grown in DMEM (high glucose; Gibco/BRL) supplemented with 10% fetal calf serum (FCS; ThermoFisher). 293F cells (ThermoFisher) were grown either as suspension cells in chemically-defined Freestyle media (ThermoFisher) in uncoated tissue culture shaker flasks on a shaking platform at 110 rpm, or in DMEM supplemented with 10% FCS in standard, coated tissue culture flasks or dishes. Cells were transfected using Lipofectamine 2000 reagent (ThermoFisher). In brief, 5 μgs of plasmid DNA was diluted into 0.3 mls of Opti-Mem medium (Gibco/BRL), while 15 μls of Lipofectamine 2000 was diluted into a separate 0.3 mls of Opti-Mem medium, and both mixtures were incubated separately for 5 minutes. The two mixtures were mixed together and incubated for a further 15 minutes. Next, growth medium was removed from a T-25 coated tissue culture flask containing HEK293 cells at ˜70-90% confluency. The cells were then washed with 5 mls of Opti-Mem medium (at 37° C.), all liquid was removed, and the 0.6 ml mixture of DNA, Lipofectamine 2000 and Opti-Mem was added to the flask. After gentle rocking to distribute the mixture across the entire flask surface, the flask was incubated for 15-20 minutes in a tissue culture incubator. The DNA/Lipofectamine 2000/Opti-MEM solution was then removed, 5 mls of DMEM+10% FCS was added to each flask, and the cells were then returned to the incubator for between 1 and 2 days, depending on the experiment. For lentiviral transductions, 1 ml of unconcentrated virus stock was added to 10 mls of culture media in the presence of 6 μg/ml polybrene and ˜3×106 cells in a 10 cm tissue culture dish, incubated for 2 days, and then washed.
Antibiotic-resistant cell lines were generated from transfected or transduced cell HEK293
cells by splitting cells onto 150 mm dishes containing DMEM, 10% FCS, and the appropriate antibiotic. Antibiotics were used at the following concentrations: 400 μg/ml G418, 20 μg/ml blasticidin, 400 μg/ml hygromycin B, 3 μg/ml puromycin, and 200 μg/ml zeocin (these concentrations approximately twice that required to kill HEK293 cells (data not shown)). Transfected cell populations were re-fed every 3-4 days until distinct, drug-resistant clones were large enough to be seen by eye and all antibiotic-sensitive cells between the drug-resistant colonies had died off, typically 10-14 days. The drug-resistant cells from each transfected population were then pooled to create polyclonal cell lines and expanded until they had grown for 4 weeks from the date of transfection in selective media. Each cell line was then processed for flow cytometry, fluorescence microscopy, and/or immunoblot.
Cells were suspended by trypsinization, washed in Hank's buffered saline solution (HBSS) and resuspended at a concentration of 1×107 cells per ml in cold (4° C.) HBSS containing 0.1% FBS. Cell suspensions were maintained on ice, diluted to a concentration of 1×106 cells per ml, and examined for tdTomato or mNeonGreen fluorescence by flow cytometry on a Beckman MoFlo Cell Sorter equipped with 355 nm, 488 nm, and 633 nm lasers set to the appropriate detection wavelength. The relative brightness was determined for thousands of individual cells in each cell line using Beckman MoFlo software and reported as scatter plots, average relative brightness, and coefficient of variation.
Cells were seeded onto sterile (autoclaved) borosilicate cover glasses in tissue culture dishes and grown overnight in normal media. The cover glasses were removed from the tissue culture dishes, washed, and fixed in 3.7% formaldehyde in Dulbecco's modified phosphate-buffered saline (DPBS), pH 7.4, for 15 minutes. The cover glasses were then washed in DPBS, incubated in DPBS containing DAPI (5 μg/ml) for 15 minutes, washed 5 times in DPBS, and mounted on a glass slide containing ˜8 μls of mounting solution (90% glycerol, 100 mM Tris pH8.5, 0.01% para-phenylenediamine). After removal of excess mounting solution, the cells were examined using a Nikon Eclipse TE200 microscope equipped with Nikon S Fluor 20x, 0.75 aperture objective and an Andor Neo sCMOS DC-152Q-COOF digital camera. Images were processed using Photoshop and assembled in illustrator (Adobe).
Equal numbers of each HEK293-derived cell line were lysed in SDS-PAGE sample buffer (20% glycerol, 4% SDS, 120 mM Tris-HCl (pH 6.5), 0.02% bromophenol blue) at room temperature, then frozen and thawed once. The thawed samples were then boiled for 10 minutes, spun at 10,000×g for 2 minutes to pellet insoluble materials, loaded onto 4-15% polyacrylamide gradient gels (Bio-Rad), and electrophoresed according to the manufacturer's suggestions. Proteins were then transferred to immobilon-P membranes (Amersham), incubated in blocking solution (5% non-fat dry milk in TBST (138 mM NaCl, 2.7 mM KCl, 50 mM Tris, pH 8.0, 0.05% Tween-20) for two hours, and then in blocking solution containing primary antibodies overnight at 4° C. (rabbit polyclonal anti-p2a antibody was used at a dilution of 1:1000 and anti-actin antibodies were used at a dilution of 1:1000). The membranes were washed 5 times in TBST and then incubated with blocking solution containing secondary antibodies conjugated with horseradish peroxidase (HRP) at a dilution of 1:5000 for 1 hour. The membranes were washed 5 times with TBST, incubated in HRP-activated chemiluminescence detection solution (Amersham ECL Western Blotting Detection Reagents; cat #RPN2106), and imaged using a GE Amersham Imager 600. Images were exported as JPEG files, analyzed using ImageJ software, and processed using Photoshop software (Adobe).
293F-derived cells were grown in sterile shaker flasks containing 80 mls of Freestyle media for 5 days at a starting concentration of 5×105 cells/ml. Clarified tissue culture supernatants (CTCSs) of each culture were then generated by centrifuging the cultures at 5000×g for 15 minutes to remove all cells, and passing the resulting supernatant through a 0.22 μm filter to remove large cell debris. ˜80 mls of the CTCS was then concentrated to ˜0.5 ml using a 100 kDa molecular weight cut-off angular filtration unit (Centricon-70) according to the manufacturer's suggestions. The resulting samples were then passed over an Izon qEV 35 nm size exclusion chromatography column using PBS, pH 7.4 as column buffer and collecting 0.5 mls fractions. Fractions 4, 5, and 6 contained exosomes (data not shown) and were pooled to generate each exosome preparation. These were examined by nanoparticle tracking analysis using a Particle Matrix ZetaView Twin 488 & 640 (PMX-220-12C-R4) according to the manufacturer's suggestions.
Heterogeneous Expression from NeoR and BsdR-Linked Transgenes
The classic approach to generating a transgenic mammalian cell line is to transfect (or transduce) the cells with a plasmid (or virus) carrying two genes, one encoding the recombinant protein of interest, and the other encoding a dominant selectable marker that confers resistance to an otherwise toxic antibiotic(4,5). The NeoR gene confers resistance to the protein synthesis inhibitor G418 (geneticin) and is the selectable marker on many if not most mammalian cell expression vectors, including pcDNA3. To document the outcomes of a classic, two-gene transgenesis experiment using the NeoR marker gene, the present inventors first created a derivative of pcDNA3 designed to encode 3×NLS-tdTomato, a form of the red fluorescent protein tdTomato(45) that carries three copies of a nuclear localization signal (NLS)(46) at its N-terminus (
HEK293 cells were transfected with this plasmid (pJM825), grown for one day in normal media, followed by transferring half of the transfected cells into G418-containing media and half into blasticidin-containing media. Thousands of antibiotic-resistant clones emerged from each selection. After two weeks, these clones were pooled to create two mixed-clone cell lines, which were then grown for an additional two weeks in antibiotic-containing media and assayed for 3×NLS-tdTomato expression by flow cytometry (
These results raised the question of whether all dominant selectable markers yield cell lines with similarly low and heterogeneous levels of recombinant protein expression. To explore this issue, the present inventors created a new set of bicistronic expression vectors in which 3×NLS-tdTomato expression was linked via the p2a peptide to the NeoR(3), BsdR(29), HygR(27), PuroR(26), and BleoR(28) markers (
22%
The NeoR- and BsdR-resistant cell lines displayed the lowest average relative brightness and high degrees of cell-to-cell variation in 3×NLS-tdTomato fluorescence (458, with a coefficient of variance (c.v.)=103; and 522, with c.v.=82, respectively). In contrast, the HygR- and PuroR-based cell lines displayed higher and more homogeneous levels of 3×NLS-tdTomato expression (794, c.v.=62; and 803, c.v.=44, respectively), and the BleoR-based cell line displayed the highest and most homogeneous expression of 3×NLS-tdTomato (1754, c.v.=46).
To determine whether these effects were specific to 3×NLS-tdTomato or could be extrapolated to other recombinant proteins, the present inventors created another set of vectors designed to express an exosomal cargo protein, CD81(48), an integral plasma membrane protein that is highly enriched in exosomes(49). Moreover, the present inventors expressed CD81 as a fusion protein with the green fluorescent protein mNeonGreen(50), allowing the detection of this protein (CD81mNG) by fluorescence-based techniques (
Taken together, the preceding results indicate that each selectable marker and antibiotic establish a distinct threshold of transgene expression below which no cell can survive, with the most permissive markers (NeoR and BsdR) allowing survival at low, medium or high levels of transgene expression, the most restrictive marker (BleoR) killing all but the most highly expressing transgenic cells, and cells selected using the HygR or PuroR markers falling somewhere in between. If this hypothesis is correct, similar results should be observed in orthogonal measurements of CD81mNG expression. The present inventors therefore processed these five cell lines for fluorescence microscopy, staining each sample with DAPI to stain the cell nuclei. The resulting images show that CD81mNG expression was lowest in the G418-resistant and blasticidin-resistant cell lines, higher in the hygromycin-resistant and puromycin-resistant cell lines, and highest in the zeocin-resistant cell line (
CD81 is among the most highly enriched exosomal proteins known(49) and has high potential as a carrier molecule for modifying exosome content. To determine whether its incorporation into exosomes is impacted by its level of expression in the exosome-producing cells, the present inventors transfected 293F cells (a derivative of HEK293 cells) with the PuroR- and BleoR-linked CD81mNG expression vectors described above (pC-CD81mNG*-2a-PuroR* and pC-CD81mNG*-2a-BleoR*). A day later, selection for antibiotic-resistant clones was initiated, which were expanded as pools of puromycin-resistant and zeocin-resistant cells. These two cell lines were then inoculated into chemically-defined media in shaker flasks and grown for 5 days. The cells were then removed from the conditioned media and exosomes were purified by a combination of low speed centrifugation, size exclusion filtration, filtration-based concentration, and size exclusion chromatography. The two exosome preparations were then interrogated by nanoparticle tracking analysis (NTA) using a Particle Metrix Zetaview PMX220(51) to measure the concentrations, sizes, and CD81mNG fluorescence of exosomes in each sample (
Both preparations contained large numbers of extracellular vesicles, the vast majority of which had the size expected of exosomes (mean diameters of 112 nm and 114 nm, respectively), confirming that exosomes were purified, and not the larger microvesicle class of extracellular vesicles. The puromycin-resistant cell line, which expresses high levels of CD81mNG, released a population of exosomes in which ˜25% displayed detectable levels of CD81mNG fluorescence. In contrast, the zeocin-resistant cell line, which expresses higher levels of CD81mNG, released a population of exosomes in which 70% carried detectable levels of CD81mNG, a >2-fold increase in exosome occupancy that is consistent with a stochastic model of exosome biogenesis (42).
The present inventors predicted that the most restrictive selectable markers such as BleoR and PuroR establish a high threshold of transgene expression. If this prediction is correct, then these markers should at least partly blunt the impact of other vector design variables on the levels of recombinant protein expression in polyclonal pools of antibiotic-resistant cells. To explore this possibility, the present inventors created a series of eight DNA vectors that express the identical recombinant protein (CD81mNG-2a-PuroR) from two different transcriptional control elements (CMV(9) or SFFV long terminal repeat (LTR)(8)) delivered by four distinct vector systems: non-replicating plasmids (pC and pS), Sleeping Beauty transposons (pITRSB-C and pITRSB-S)(18,19), Epstein Barr Virus (EBV)-based episomes (pREP-C or pREP-S)(22), or replication-defective, self-inactivating lentiviruses (Lenti-C or Lenti-S) (
To test whether similar results would be observed in the absence of puromycin selection, the present inventors transfected HEK293 cells with the same six DNA vectors at two days post-transfection (
These transiently-transfected cell populations contained many more non-expressing cells, a much lower average level of CD81mNG fluorescence, and much higher cell-to-cell variation of CD81mNG expression. These results indicate that the CMV-based vectors drove higher CD81mNG expression than the SFFV LTR-based vectors, especially when delivered on an EBV-based replicating vector. These results support the idea that the similarity in CD81mNG in expression seen in the eight puromycin-selected cells is due to the restrictive nature of puromycin selection following transfection with PuroR-linked transgenes. The present inventors also collected data on the time course of unselected, CMV-driven, CD81mNG expression, which confirmed that transgene expression declines rapidly in the absence of antibiotic selection from multiple gene delivery platforms.
To determine whether choice of selectable marker has a similar effect on transgene expression in other mammalian cell lines, the present inventors transfected the simian virus 40-transformed African green monkey kidney cell line COS-7 with the non-replicating plasmids designed to express CD81mNG from polycistronic ORFs linked by a 2a peptide to the NeoR(3) BsdR(29), HygR(27), PuroR(26), and BleoR(28) markers (
The creation of transgenic mammalian cells is a critical step in many biomedical research projects. However, there is no simple, inexpensive, and rapid method for generating transgenic cell lines that express high and relatively homogeneous levels of linked recombinant proteins. The present inventors explored herein the impact that the choice of selectable marker has on the levels of a linked recombinant protein, and found that it can have up to a 10-fold effect on expression level. Moreover, the present inventors established that the choice of selectable marker also has a pronounced effect on the cell-to-cell variation in transgene expression, with the highest variation correlating with the lowest average expression and the lowest cell-to-cell variation observed in the highest-expressing polyclonal cell lines.
The simplest interpretation of these observations is that each selectable marker-antibiotic pair establishes a threshold of transgene expression below which no cell can survive. The present inventors anticipate that these thresholds are determined, at least in part, by each marker protein's mechanism of action, intrinsic activity, and stability within the cell. Given that these variables are likely to be distinct for nearly all proteins, it is not surprising that each marker/antibiotic pair established a distinct threshold of transgene expression. More specifically, this model predicts that highly efficient and long-lived selectable marker proteins inactivate their cognate antibiotic even at very low levels of expression. As a result, their use results in the survival of cells that express almost any level of the linked recombinant protein, which manifests in pooled, polyclonal cell lines as a low average level of transgene expression and a high degree of cell-to-cell variation in transgene expression levels. These properties correspond relatively well to those observed for cell lines generated using the NeoR or BsdR markers, indicating that the NeoR and BsdR selectable marker enzymes may be highly active, stable, or both. As for the higher levels of transgene expression in hygromycin-resistant or puromycin-resistant cell lines, the present inventors posit that the HygR and PuroR enzymes may be less active, less stable, or both. And finally, the fact that the BleoR marker consistently yielded cell lines with the highest and least heterogeneous levels of transgene expression indicates that it has the lowest activity of all selectable marker proteins, consistent with its non-catalytic mechanism of zeocin inactivation. This interpretation of our results is actually consistent with the known mechanism of BleoR-mediated resistance to zeocin, which is non-catalytic and involves its chelation of zeocin at a 1:1 molar ratio(52).
A major outcome of this study is the realization that the BleoR marker can be used to quickly create polyclonal cell lines expressing relatively high and homogeneous levels of a linked recombinant protein. Although the data presented herein establishes this for only two proteins, follow-on studies indicate that it works for many other proteins (e.g. EGFR, PD-L1, HEMO, etc.). While the levels of expression attained by this system may still be less than those achieved by more labor-intensive approaches, the present approach to recombinant protein expression is significantly faster and less time consuming, and clearly superior to the traditional, two-gene strategy for making transgenic mammalian cells. As for how to make best use of the expression system described herein, the present inventors contemplate transfecting a very large population of cells (>1×106), in part because the zeocin selection kills all but the most highly expressing transgenic clone, and in part because the number of initial zeocin-resistant clones determines the time required to generate a working cell line. That being said, use of the BleoR marker and zeocin is not free of concern, as zeocin kills cells by binding DNA and inducing DNA damage(54). In fact, zeocin-induced DNA damage can even occur in BleoR-expressing, zeocin-resistant cell lines(55). While the PuroR-derived cell lines displayed lower levels of transgene expression, the present inventors are unaware of long-term damage associated with extended growth of PuroR-derived cell lines in its cognate antibiotic, and the same can be said for HygR/hygromycin-based selection of transgenic cell lines.
These findings are also relevant to the creation of clonal transgenic cell lines. After all, they represent strong evidence that use of the BleoR marker eliminates cells that express low or medium levels of a linked recombinant protein, with similar but lesser effects attained by use of the PuroR or HygR markers. Similar considerations recommend use of the BsdR marker for projects that require a low level of transgene expression, as the cell lines generated using the BsdR marker and blasticidin selection allowed survival of poorly-expressing cell clones. As for the NeoR marker, the present inventors cannot recommend its use for any purpose, largely because the NeoR-derived cell lines displayed the greatest variation in transgene expression, contained the highest percentage of non-expressing cells, and exhibited time-dependent changes in recombinant protein expression. Taken together, these considerations raise the possibility that the NeoR gene, which has been used more often than any other selectable marker (see addgene.org), may in fact be the least useful of all.
These studies were performed primarily in the context of expressing CD81, an exosomal protein. The present inventors did this because they wanted to know whether their findings related to transgene expression might inform their approach to exosome engineering. A variety of observations support the hypothesis that exosome biogenesis is essentially a stochastic process in which the content of any individual exosome is determined by the local concentrations of exosome cargo molecules in the vicinity of a nascent vesicle budding event(42). Under this model, increasing the expression of an exosomal cargo protein within the cell should lead to its presence on a higher percentage of exosomes. Consistent with this prediction, the present inventors observed that 293F cells expressing CD81mNG from a PuroR-linked transgene, which selects for high transgene expression, released a population of exosomes in which ˜25% of the vesicles contained mNeonGreen fluorescence. In contrast, 293F cells expressing CD81mNG from a BleoR-linked transgene, which selects for significantly higher transgene expression, released a population of exosomes in which ˜70% contained mNeonGreen fluorescence, an ˜2.5-fold increase in exosome occupancy by this engineered cargo molecules. Taken together, these observations indicate that the choice of selectable marker is an important consideration in the genetic modification of exosome content, and is therefore relevant to the production of exosome-based therapies, standards and controls.
The general relevance of the present findings can only be determined by testing this system in the context of other recombinant proteins, other cell lines, and other research environments. To facilitate this process the present inventors have created a suite of vectors that are designed to drive the bicistronic expression of recombinant proteins in frame with (a) thep2a peptide and (b) the NeoR, BsdR, HygR, PuroR, or BleoR coding sequences, transcribed from either the CMV enhancer/promoter or the SFFV LTR, and carried on simple plasmids, Sleeping Beauty transposons, EBV-based episomes, or lentiviral vectors (
The presented inventors generated Sleeping Beauty transposons in which the CMV promoter was used to drive the expression of bicistronic open reading frames (ORFs) encoding (i) the fluorescent protein mCherry, (ii) an 18 amino acid-long viral 2a peptide (p2a), and (iii) an array of different selectable marker genes. These marker genes consisted of previously characterized, codon-optimized genes encoding the BleoR, BsdR, NeoR, HygR, and PuroR proteins, as well as forms of each that were tagged at their N-terminus with the destabilization domains (DD) from human estrogen receptor (ER50) and Escherichia coli dihydrofolate reductase (ecDHFR). (
Transfection of HEK293 cells with vectors carrying the BleoR marker, followed by selection in zeocin-containing media, generates cell lines with the highest average levels of linked recombinant protein expression. It should be noted that BleoR-based vectors also generate the lowest percentage of antibiotic-resistant clones, for the very same reason, which is that only those transgenic cell lines with the very highest levels of transgene expression are able to survive. When the present inventors transfected cells with vectors carrying degron-tagged forms of the BleoR gene, the number of clones was even lower for the ER50BleoR and none were obtained for cell transfected with the vector carrying the ecDHFRBleoR marker. As for the BleoR-selected and ER50BleoR-selected cell lines that were generated following selection, flow cytometry revealed that degron tagging the BleoR marker resulted in an ˜2.5-fold increase in transgene expression. Specifically, it was observed that the mean mCherry fluorescence brightness increased from 16,024 (arbitrary units) in the BleoR cell line to 37,141 in the ER50BleoR cell line (
In the above analysis of the effect of selectable marker choice on transgene expression levels, the present inventors demonstrated that it could have as much as a 10-fold effect on the expression levels of a linked recombinant protein. The present inventors show here that this effect can be even larger, as much as 28-fold, which was the difference in mean mCherry brightness that was observed for HEK293 cells generated using the BsdR marker as compared to those generated using the ER50BleoR marker (1,308 vs 37,141;
For most of the past 40 years, the NeoR gene was the most commonly used dominant selectable marker in mammalian cell transgenesis. However, the present inventors' study establishing that choice of selectable marker had a pronounced effect on transgene expression levels revealed that the NeoR gene was one of the two most poorly-performing selectable marker genes, routinely yielding cell lines with the lowest average levels of transgene expression and the highest degree of cell-to-cell variability in transgene expression. Degron tagging did not improve this marker by much, as the highest levels of linked recombinant protein expression, observed for cells selected with the ecDHFRNeoR marker, were at best 30% higher than in cells selected with the NeoR marker (
Degron tagging alone had even less of an effect on HygR performance characteristics, with no improvement observed for ER50HygR and only 20% improvement observed for ecDHFRHygR (
Degron tagging was also applied to the PuroR gene, but it was far less effective than what was observed for the BleoR or BsdR genes. Specifically, flow cytometry experiments revealed that cell lines generated using the ER50 and ecDHFR destabilization domains expressed ˜70% higher expression of the linked recombinant protein (
The dominant selectable markers that form the foundation of the aforementioned studies were all cloned decades ago at a time when the utility of a potential selectable marker gene was thought to be proportional to the numbers of antibiotic-resistant clones that were obtained following a standard transfection and selection protocol. This property of yielding large numbers of antibiotic-resistant clones, is associated with low average transgene expression and high cell-to-cell variability in transgene expression. The present inventors therefore asked whether homology probing could be used to identify new selectable marker proteins that have better performance characteristics than our existing selectable marker genes. Specifically, the present inventors identified a homolog of the PuroR gene (
The present inventors hypothesized that each dominant selectable marker establishes a threshold of transgene expression below which no cell can survive, that each marker gene/protein establishes a distinct threshold of transgene expression, and that this threshold is determined by numerous properties of the marker protein, some of which are intrinsic to the protein (e.g. binding affinity for the antibiotic, mechanism of antibiotic inactivation, etc.) while others are extrinsic (e.g. its ability to fold, function and avoid proteolysis in the alien environment of the mammalian cell). Here the present inventors tested a key prediction of this hypothesis by creating transgenes in which the destabilization domains (DDs) from the human estrogen receptor (ER50) and the E. coli DHFR were appended to the N-terminus of the BleoR, PuroR, HygR, BsdR, and NeoR marker proteins, transfecting these transgenes into 293F cells, selecting for antibiotic-resistant cell lines, then measuring the levels of the linked upstream recombinant protein (mCherry) in the resulting mixed-clone cell lines. In each case, it was observed that cell lines generated with degron-tagged marker proteins expressed higher and more homogeneous levels of mCherry expression. Moreover, it was observed that the extent of this effect was, in general, slightly greater for ecDHFR-tagged markers than for ER50-tagged markers.
These experimental results describe significantly improved selectable markers for each of the five antibiotics used in mammalian cell transgenesis experiments. BleoR, which was already the best marker for selecting transgenic cell lines with high levels of transgene expression, was improved by >2-fold by addition of the ER50 domain, making ER50BleoR the single best marker for selecting cell lines with high levels of transgene expression. The next most significant development was the PuroR2 marker, use of which allows puromycin to select for cells with the second-highest level of transgene expression, slightly higher than BleoR and ˜2.5-fold higher than PuroR. Even larger increases of 5- to 6-fold were observed for degron-tagged forms of BsdR, as ER50BsdR- and ecDHFRBsdR-selected cells displayed levels of transgene expression similar to that of cells carrying the PuroR or HygR markers, and higher than those selected with any of the three forms of the NeoR marker. As for the degron-tagged forms of the HygR and NeoR proteins, they also yielded cell lines with higher levels of transgene expression, though the effects were in all cases quite modest (<30% increase). In short, this study has generated improved markers for all five major mammalian cell antibiotics, and in particular ER50BleoR for zeocin selection, PuroR2 for puromycin selection, and both ER50BsdR and ecDHFRBsdR for blasticidin selection.
The present inventors have found that DD-stabilizing drugs alter the relationship between SM function and transgene expression. More specifically, the ER50 DD is stabilized by adding 4-hydroxytamoxifen (4-OHT) to the culture media, resulting in a higher number of initial antibiotic-resistant cell clones, with reduced average level of transgene expression. However, once established, these antibiotic-resistant cell lines can be passaged in progressively lower concentrations of the stabilizing drug, eventually yielding cell lines that grow well in the absence of 4-OHT. The same is true for cell lines selected with ecDHFR DD-tagged marker, with the exception that trimethoprim is used instead of 4-OHT. The result is that the DD-tagged markers can be used with a sliding scale of stabilizing drug to achieve the desired level of transgene expression.
Human cells do not have a natural, endogenous, functional DNA transposon. However, three different transposons have been developed for use in human and other mammalian cells: Sleeping Beauty, PiggyBac, and TcBuster. The present inventors chose Sleeping Beauty (SB) transposons as the platform for transgene delivery. To implement use of SB transposons, the present inventors synthesized the plasmid pITRSB as shown in
The critical features of this plasmid are (i) a gene (e.g., RSV-SB100x-pAn) designed to express an optimized version of the SB transposase enzyme, located outside the transposon and (ii) a functional transposon comprised of the inverted tandem repeats (ITR-L and ITR-R) that define the left and right ends of the transposon (the transposon is comprised of all DNA between the 5′most bp of the ITR-L to the 3′most bp of the ITR-R).
This vector works as follows:
The present inventors used this pITRSB vector platform to deliver an exosomal marker-expressing transgene (CMV-CD81-mNeonGreen-2a-PuroR), validating that the system worked as planned. These experiments also revealed that transgene delivery via SB transposition led to higher levels of expression than either plasmid-based delivery or lentivirus-based delivery.
To determine whether transposon-mediated transgene delivery was refractory to transgene fragmentation, the present inventors created derivatives of pITRSB designed to express multiple genes. However, the expression of SM-expressing genes required some consideration for how best to express them as separate, stand-alone genes.
The present inventors engineered the SM-expressing gene to be driven from the weak, minimal EF1alpha promoter (EFS), while the reporter gene for monitoring transgene delivery—mCherry—was expressed from the strong CMV promoter.
pS179 was transfected into 293F/tet1 cells (to be described below) followed by addition of 3 microgram/ml puromycin. The resulting colonies, of which there were thousands, were pooled, expanded, and assayed by flow cytometry (
The clear separation of pS179 transfected cell line from the 293F cell line as shown in
While it is possible to achieve high-level expression of constitutively expressed transgenes, there are many instances when high-level constitutive expression is either undesired or impossible. To overcome this limitation the present inventors sought to develop Tet-On derivatives of 293F cells which would allow for tetracycline/doxycycline-inducible expression of transgenes under the control of tet-regulated promoters (e.g. TRE3G).
To pursue this strategy the present inventors combined (A) the existing knowledge of Tet-On gene expression systems, which shows that the rtTAv16 protein displays higher Tet-regulated gene expression than any other Tet-regulated transcription factor, with (B) the information the present inventors had learned regarding the effect of selectable marker (SM) on transgene expression as described above, specifically:
These considerations led the present inventors to create the plasmid designated as “pS147” (
Following transfection of cells with this plasmid and selection for zeocin-resistant cells, the rtTAv16 protein will be expressed as a 2a-tagged protein, and thus detectable using anti-2a antibodies (the 2a expression system always appends the first 17 amino acids of the 2a peptide to the C-terminus of the upstream protein). These cell lines are designated with the generic term of “Ftet” lines, and these lines are further segregated according to whether the cell line was generated by pooling numerous ZeoR clones (designated as “Ftet1”), or by picking single cell clones (SCCs) and screening them for relative expression of the rtTAv16-2a protein, with the highest-expressing SCC designated as the cell line “Ftet2”.
In the course of these experiments it was observed that TRE3G-regulated transgenes displayed a pattern of stochastic, doxycycline-independent expression in Ftet cells, in some cases leading to significant transgene expression.
The present inventors created the plasmid designated as “pCG210”, which is identical to pS147 except that it encodes rtTAv16/G72P with a single amino acid substitution. pCG210 was transfected into 293F cells, selected in zeocin-containing media, followed by (a) pooling of thousands of cell clones to make the Ftet3 cell line, and (b) cloning of the 10 fastest-growing single cell clones (SCCs).
In addition, the present inventors combined the ER50BleoR marker with both rtTAv16/G72 and rtTAv16 to create two additional vectors (designated as “pCG211” and “pCG212” designed to drive even higher-level expression these transcription factors. These plasmids were transfected into 293F cells followed by selection in zeocin-containing media supplemented with 4-hydroxytamoxifen to stabilize the ER50 degron. Once again, pooled cell lines and single cell clones were derived from each of these transfections.
To assess the levels and homogeneity of rtTAv16-2a or rtTAv16/G72P-2a in these various cell lines, the present inventors processed them for immunofluorescence microscopy using antibodies specific for the viral 2a peptide epitope tag. These cell lines varied significantly in their levels of linked protein expression (
Based on the above developments, the present inventors created and validated a vector platform that combines: (a) engineered selectable marker genes that drive high-level recombinant protein expression; (b) Sleeping Beauty transposition, which delivers transgenes at high efficiency; and (c) a doxycycline-inducible transgenes, using a promoter (TRE3G) that can drive high-level, rtTA-mediated, dox-inducible transgene expression.
These features are evident in the plasmid designated as “pS180” (
To validate its utility for driving high-level, doxycycline-inducible transgene expression, the present inventors transfected p5180 into Ftet1 cells, selected for puromycin-resistance, pooled all colonies to form a single, mixed-clone cell line, and then assayed mCherry expression by flow cytometry after 1 day of culture in normal media or media supplemented with 100 ng/ml doxycycline (
In the course of these experiments, the present inventors asked whether the CMV enhancer/promoter might also be inducible, not by doxycycline but by existing signaling pathways that can be activated in 293F cells. In particular, it was found that the CMV enhancer has a number of binding sites for the transcription factors NFAT and NF-kB, a pair of transcription factors that are known to be activated by protein kinase C (PKC). Furthermore, the natural product prostratin is a potent activator of conventional PKC isoforms. To assess its ability to drive higher levels of transgene expression from the CMV promoter, the present inventors assayed mCherry expression by flow cytometry in Ftet1 cells grown in the presence or absence of prostratin (
These results indicate that addition of prostratin elevates mCherry expression nearly 2-fold. By deduction, these results indicate that addition of prostratin might also elevate expression of the rtTAv16 protein in Ftet cells, as the expression of the rtTAv16 protein in these cell lines is under the control of the CMV promoter. Furthermore, since the Tet-regulated mCherry gene in Ftet1/pS180 cells shows detectable levels of dox-independent expression (
The resulting data establish that addition of prostratin alone elevates mCherry expression ˜2-fold, and that addition of prostratin further increases doxycycline-induced gene expression by ˜15%.
The present inventors next tested whether the choice of selectable marker affected the levels of transgene expression from Sleeping Beauty transposons. Using the TRE3G-mCherry transgene as reporter, the PuroR coding region in the EFS-PuroR gene was replaced with the ER50PuroR and ecDHFRPuroR markers, the resulting plasmids were transfected into Ftet1 cells, and then the cells were placed in puromycin-containing media, in some cases supplemented with appropriate concentrations of 4-hydroxy-tamoxifen (4OHT) or trimpethoprim, drugs that stabilize the ER50 and ecDHFR destabilization domains, respectively (transfection with the plasmid carrying the EFS-ecDHFRPuroR marker gene yielded no puromycin-resistant colonies either in the presence or absence of trimethoprim).
Transfection with the plasmid carrying the EFS-ER50PuroR marker yielded colonies in the presence of 4OHT but not in its absence. These selections were carried out at 1 microgram/ml puromycin. Cells transfected with the PuroR-based plasmid were selected at both 1 and 3 microgram/ml puromycin. The three cell lines arising from these transfections were then cultured in the presence of 100 ng/ml doxycycline for 24 hours and assayed by flow cytometry (
These data support the present inventors' operating hypothesis of selectable marker function. Moreover, these data show that elevating antibiotic concentration elevates the threshold of transgene expression needed for antibiotic resistance, though only slightly. It should also be noted that these mCherry expression levels reflect leaky expression from the TRE3G promoter in the absence of doxycycline and are therefore likely reporting on differences in gene dosage that are selected by each combination of selectable marker and antibiotic concentration.
To explore these conclusions further, the present inventors created a series of vectors using the NeoR and BsdR markers and their degron-tagged derivatives. In brief, these experiments revealed, once again, the effect of degron tagging on transgene expression, as 293F/Ftet2 cells selected with the NeoR marker showed ˜1.5-fold higher mCherry expression when grown in a 2x concentration of G418 (800 μg/ml), and Ftet2 cells selected with the ER50NeoR gene in the absence of 4OHT showed mCherry expression levels twice again as high (
The above results support the hypothesis that cell and exosome engineering can be best achieved using Sleeping Beauty-based gene delivery and doxycycline-induced expression of recombinant proteins of interest.
Based on the above information, the present inventors generated a single Sleeping Beauty transposon carrying 5 genes, one that expresses the PuroR protein from a crippled EF1alpha promoter, and 4 other genes in which codon-optimized versions of the S, N, M, and E ORFs are driven from the dox-inducible TRE3G promoter (
This plasmid, designated as “pS226”, also carries 4 amino acid substitution mutations that are designed to prevent furin-mediated cleavage at the S1/S2 junction site, and two additional amino acid substitution mutations that are believed to stabilize the trimeric form of the Spike protein.
The plasmid pS226 was transfected into Ftet2 cells, a single cell clone that was generated by transfection of 293F cells with the plasmid pS147 and which grows in media containing 200 ug/ml zeocin. Two days later, half the cells were placed in media containing 1 μg/ml puromocyin and half were placed in media containing 3 μg/ml puromycin. No colonies arose on the plates fed with the higher concentration of puromycin, but many clones arose on cells fed with media containing 1 μg/ml puromycin.
These puromycin-resistant clones were pooled and expanded leading to the first master cell bank. In test experiments, addition of doxycycline to the culture media led to expression of all 4 proteins, as well as the appearance of all 4 proteins in exosome/VLP fraction generated by either of two purification protocols (differential centrifugation and filtration/chromatography, respectively).
For VLP production, cells carrying the S/N/M/E-expressing transposons are resuspended in freestyle media supplemented with 800 ng/ml doxycycline, prostratin, and forskolin at a density of 0.5-1×10{circumflex over ( )}6 cells/ml and cultured with shaking for 3 days.
For VLP purification, the culture media was spun at 500×g for 5 minutes, the SN transferred to a fresh tube, spun again at 5000×g for 10 minutes, followed by gravity-flow filtration of the SN through a 0.22 micron pore size PES filter sterilization unit (125, 250, or 500 ml capacity for large surface area filtration). The filtrate, which is referred to as a clarified tissue culture supernatant (CTCS) was then concentrated using a Centricon 70 spin concentrator with a 100 kDa pore size cutoff. Thus, starting CTCS of 70, 140, or 210 mls can be reduced to a volume of 0.5 ml. This material is then loaded on qEV size exclusion chromatography column (Izon) and 0.5 mls fractions are collected. Fractions 1-3 are the void volume, fractions 4-6 contain the VLPs, and subsequent fractions contain proteinaceous contaminants. The resulting 1.5 mls are pooled and assayed for protein concentration and numbers of and sizes of vesicles in the sample.
The resulting VLP preps can be used for biological studies of virus-host interaction, but also as the key material of a vaccine.
The present inventors also generated a plasmid designated “pS225”, which is identical to pS226, except for the fact that pS226 expresses the Wuhan-1 strain Spike protein, and a plasmid designated “pCG201”, which is identical to pS226, except for the fact that pCG201 expresses the D614G Spike protein (only 1 amino acid difference from the Wuhan-1 strain Spike protein). SARS-CoV-2 VLPs were made by transfection of the 293F/pS147 SCC with pS225, pS226, or pCG201, each of which are designed for transposon-mediated delivery of dox-regulated genes expressing the SARS-CoV-2 S, N, M, and E proteins.
F/Tet-on Cell Lines that Make SARS-CoV-2 VLPs: Expression Data (Immunoblot)
The two cell lines (293F/pS147/pS225 and 293F/pS147/pS226) were suspended in Freestyle media in the presence of 100 ng/ml dox, incubated in TC shaker flasks for 3 days, with aliquots removed at d0 and d2. Cell lysates=0d, 2d, and 3d lanes. Exosomes/VLPs were collected from the TC SN at d3. Immunoblots (
F/Tet-on Cell Lines that Make SARS-CoV-2 VLPs: Ultrastructural Analysis
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application. Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 63/155,268, filed Mar. 1, 2021; U.S. Provisional Patent Application Ser. No. 63/110,325, filed Nov. 5, 2020; U.S. Provisional Patent Application Ser. No. 63/108,847, filed Nov. 2, 2020; U.S. Provisional Patent Application Ser. No. 63/061,766, filed Aug. 5, 2020; U.S. Provisional Patent Application Ser. No. 63/000,211, filed Mar. 26, 2020; U.S. Provisional Patent Application Ser. No. 62/990,946, filed Mar. 17, 2020; and U.S. Provisional Patent Application Ser. No. 62/989,525, filed Mar. 13, 2020, the contents of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/022200 | 3/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63155268 | Mar 2021 | US | |
| 63110325 | Nov 2020 | US | |
| 63108847 | Nov 2020 | US | |
| 63061766 | Aug 2020 | US | |
| 63000211 | Mar 2020 | US | |
| 62990946 | Mar 2020 | US | |
| 62989525 | Mar 2020 | US |
