Patent Application
20220010335
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “109007-23530US01_ST25.txt” created on Aug. 7, 2020 and is 2,079 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates generally to protein and nucleic acid delivery components, compositions, mechanisms and methods of delivery thereof.
Delivery of genes and proteins into target cells would be essential for mechanistic research and the prevention and treatment of many human diseases such as cancer, infectious diseases and genetic diseases. However, formulating a vehicle that can efficiently and safely deliver both genes and proteins into the target cell is still a major challenge. The present application overcomes the shortcomings of the prior art as described herein.
According to a first broad aspect, the present invention provides a product comprising a hybrid viral vector (e.g. T4-AAV) comprising: a first virus (e.g. bacteriophage T4); one or more second virus (e.g. adeno-associated virus, AAV) attached to the head of the prokaryotic virus through cross-bridges (e.g. avidin-biotin cross-bridges).
According to a second broad aspect, the present invention provides a method comprising the following steps: designing an avidin-biotin cross-bridges; attaching one end of the cross-bridge (Soc or Hoc) to the first virus (bacteriophage T4); and attaching the other end of the cross-bridge (avidin) to the second virus (AAV).
According to a third broad aspect, the present invention provides a method comprising the following steps: exposing cells of a biological specimen to a hybrid viral vector (T4-AAV) thereby bind the vector to the cells or administering the hybrid vector to a biological specimen, wherein the vector comprises one or more molecules of nucleic acid packaged in either the first or second vector or both, wherein there are optionally one or more Soc-fused proteins displayed on the surface of the first virus, and wherein the vector is internalized within the cells, wherein internalization of the vector within the cells causes the release of the packaged nucleic acid into the cytosol of each of the cells, wherein the release of the packaged one or more molecules of nucleic acid into the cytosol of each of the cells causes entry of the one or more molecules of nucleic acid into the nucleus of each of the cells, wherein the entry of the one or more molecules of nucleic acid into the nucleus of each of the cells causes transcription of the DNA or reverse transcription of the RNA and over-expression of protein(s) encoded by the nucleic acid.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.
The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments of the invention, and, together with the general description given above and the detailed description given below, serve to explain the features of the invention.
Where the definition of terms departs from the commonly used meaning of the term, applicant intends to utilize the definitions provided below, unless specifically indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood to which the claimed subject matter belongs. In the event that there is a plurality of definitions for terms herein, those in this section prevail. All patents, patent applications, publications and published nucleotide and amino acid sequences (e.g., sequences available in GenBank or other databases) referred to herein are incorporated by reference. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.
For purposes of the present invention, the term “comprising”, the term “having”, the term “including,” and variations of these words are intended to be open-ended and mean that there may be additional elements other than the listed elements.
For purposes of the present invention, directional terms such as “top,” “bottom,” “upper,” “lower,” “above,” “below,” “left,” “right,” “horizontal,” “vertical,” “up,” “down,” etc., are used merely for convenience in describing the various embodiments of the present invention. The embodiments of the present invention may be oriented in various ways. For example, the diagrams, apparatuses, etc., shown in the drawing figures may be flipped over, rotated by 90° in any direction, reversed, etc.
For purposes of the present invention, a value or property is “based” on a particular value, property, the satisfaction of a condition, or other factors, if that value is derived by performing a mathematical calculation or logical decision using that value, property or other factor.
For purposes of the present invention, it should be noted that to provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about.” It is understood that whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including approximations due to the experimental and/or measurement conditions for such given value.
For purposes of the present invention, the term “bind,” the term “binding” and the term “bound” refers to any type of chemical or physical binding, which includes but is not limited to covalent binding, hydrogen binding, electrostatic binding, biological tethers, transmembrane attachment, cell surface attachment and expression.
For purposes of the present invention, the term “vector”, “vehicle”, and “nanoparticle” are used interchangeably. These terms refer to a virus or a hybrid viral particle that can be used to deliver genes or proteins.
For purposes of the present invention, the term “tropism” refers to the tendency of a virus to infect a particular host cell or tissue.
For purposes of the present invention, the term “biological sample” and the term “biological specimen” refers to either a part or the whole of a human, vertebrate animal, invertebrate animal, microbe or plant in vitro or in vivo. The term includes but is not limited to material of human, vertebrate animal, invertebrate animal, microbe or plant origin such as human, animal, microbial or plant tissue sections, cell or tissue cultures, suspension of human, vertebrate animal, invertebrate animal, microbial or plant cells or isolated parts thereof, human or animal biopsies, blood samples, cell-containing fluids and secretion.
For purposes of the present invention, the term “capsid” and the term “capsid shell” refers to the protein shell of a virus comprising several structural subunits of proteins. The capsid encloses the nucleic acid core of the virus.
For purposes of the present invention, the term “nucleic acid” refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art. The term should be understood to include, as equivalents, analogs of either DNA or RNA made from nucleotide analogs. The term should also be understood to include both linear and circular DNA. The term as used herein also encompasses cDNA, that is complementary, or copy, DNA produced from an RNA template, for example by the action of reverse transcriptase.
For purposes of the present invention, the term “immune response” refers to a specific response of the immune system of a biological specimen to antigen or immunogen. Immune response may include the production of antibodies and cellular immunity.
For purposes of the present invention, the term “immunity” refers to a state of resistance of a biological specimen to an infecting organism or substance. It will be understood that an infecting organism or substance is defined broadly and includes parasites, toxic substances, cancer cells and other cells as well as bacteria and viruses.
For purposes of the present invention, the term “immunization conditions” refers to factors that affect an immune response including the amount and kind of immunogen or adjuvant delivered to a biological specimen, method of delivery, number of inoculations, interval of inoculations, the type of biological specimen and its condition. “Vaccine” refers to pharmaceutical formulations able to induce immunity.
For purposes of the present invention, the term “immunization dose” refers to the amount of antigen or immunogen needed to precipitate an immune response. This amount will vary with the presence and effectiveness of various adjuvants. This amount will vary with the biological specimen and the antigen, immunogen and/or adjuvant but will generally be between about 0.1 μg/ml or less and about 100 μg per inoculation.
For purposes of the present invention, the term “neck protein” and the term “tail protein” refers to proteins that are involved in the assembly of any part of the necks or tails of a virus particle, in particular bacteriophages. Tailed bacteriophages belong to the order Caudovirales and include three families: The Siphoviridae have long flexible tails and constitute the majority of the tailed viruses. Myoviridae have long rigid tails and are fully characterized by the tail sheath that contracts upon phage attachment to bacterial host. The smallest family of tailed viruses are podoviruses (phage with short, leg-like tails). For example, in T4 bacteriophage gp10 associates with gp11 to forms the tail pins of the baseplate. Tail-pin assembly is the first step of tail assembly. The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Once the heads are packaged with DNA, the proteins gp13, gp14 and gp15 assemble into a neck that seals of the packaged heads, with gp13 protein directly interacting with the portal protein gp20 following DNA packaging and gp14 and gp15 then assembling on the gp13 platform. Neck and tail proteins in T4 bacteriophage may include but are not limited to proteins gp6, gp25, gp53, gp8, gp10, gp11, gp7, gp29, gp27, gp5, gp28, gp12, gp9, gp48, gp54, gp3, gp18, gp19, gp13, gp14, gp15 and gp63.
For purposes of the present invention, the term “purified” refers to the component in a relatively pure state—e.g. at least about 90% pure, or at least about 95% pure or at least about 98% pure.
While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention.
Delivery of genes and proteins into target cells forms would be essential for mechanistic research and the prevention and treatment of many human diseases such as cancer, infectious diseases and genetic diseases. Viral vectors have been exploited as a vehicle of delivering genes and proteins into target cells. Commonly used viral vectors include eukaryotic viral vectors and prokaryotic viral vectors.
Eukaryotic viral vectors such as adenoviruses, adeno-associated viruses (AAV), retroviruses and/or lentiviruses have naturally evolved to deliver their genomes efficiently into mammalian cells, making them capable of delivering genes into human cells. However, eukaryotic viral vectors such as AAV are not efficient delivery vehicle, because of their limited delivery capacity: (1) they can only deliver one or two genes and 4-8 kb in size; and (2) they are not suitable for delivering proteins. Furthermore, their tropism to and toxicity for human cells, pre-existing immunity, and safety concerns restrict their broad application2.
Prokaryotic viral vectors, such as bacteriophage (phage) T4, overcome the drawbacks of eukaryotic viral vectors4-13. However, phages are also not efficient delivery vehicles because they lack natural mechanisms to enter mammalian cells or reach appropriate intracellular compartments following entry.
In one embodiment, the present invention provides a T4-AAV hybrid viral vector by attached AAV to a T4 head through a biotin-avidin bridge. As shown in
In one embodiment, the present invention provides a method of producing the Soc-bridged T4-AAV hybrid viral vector, comprising steps: (1) biotinylation of Soc protein; (2) attaching the biotinylated Soc (Soc-biotin) protein to T4 head to produce T4-Soc-biotin; (3) attaching avidin to T4-Soc-biotin to produce T4-Soc-avidin (TSA); (4) biotinylation of AAV; (5) attaching biotinylated AAV to TSA by binding the biotin on AAV to avidin.
In another embodiment, the present invention provides a method of producing the Hoc-bridged T4-AAV hybrid viral vector, comprising steps: (1) attaching biotin acceptor peptide (BAP) to Hoc protein to produce Hoc-BAP; (2) biotinylation of Hoc-BAP to form HBB; (2) attaching avidin to HBB to form HBBA; (3) attaching HBBA to T4 head to produce THA; (4) biotinylation of AAV; (5) attaching biotinylated AAV to THA by binding the biotin on AAV to avidin.
Next, Soc-biotin is attached to the T4 head. As shown in
Subsequently, avidin is attached to biotinylated Hoc-BAP (HBB) 328, forming HBBA. HBBA is then attached to T4 to efficiently form THA 912 (
To attach AAV to T4, AAV-DJ, virus particles were first biotinylated by activation with biotin-NHS. AAV-DJ contains three AAV capsid proteins, VP1, VP2, and VP3. The biotinylation of all three AAV capsid proteins is confirmed as shown in
The biotinylated AAV-DJ particles are then attached to the TSA and THA bridges through the binding of avidin and the biotin on AAV-DJ particles (
The conjugation of AAV to T4 is also monitored through the amount of unbound AAV. After increasing ratios of bioAAV(GFP) added to TSA or THA, unbound bioAAV(GFP) is collected and transduced into HEK293 cells to have the GFP gene carried by bioAAV expressed. Thus, the fluorescence intensity is an indication of the amount of GFP expression and the amount of unbound AAV.
Genes are Remarkably More Efficiently Delivered by T4 when AAV is Attached
It is previously shown that transduction into mammalian cells (HEK293) by T4 heads alone is very inefficient10. The present invention compared the delivery efficiency of T4 heads along and T4-AAV when the genes are packaged in T4.
In one embodiment, plasmids containing luminance genes are packaged in T4 capsid with AAV attached through TSA bridges, and delivered to HEK293 cells. The delivery efficiency is measured by relative luminescence unit.
As shown in
In one preferred embodiment, luciferase DNA is delivered into cells using T4(luci)-Soc-AAV. The delivery efficiency is indicated by relative luminescence unit.
In another preferred embodiment, luciferase DNA is delivered into cells using T4(luci)-Hoc-AAV.
An advantage of the T4-AAV hybrid vector is that both T4 and AAV can be simultaneously used to deliver genes. T4 delivers genes in the form of double-stranded DNA23, whereas AAV delivers genes in the form of single-stranded DNA, the natural state of the packaged AAV genome15.
In one embodiment, mCherry DNA (red fluorescence) is packaged into T4 capsid and GFP DNA (green fluorescence) is packaged into AAV, and both are conjugated through the Soc bridges. Then, these T4(mCherry)-AAV(GFP) nanoparticles are added to HEK293 cells for delivery and expression both green and red fluorescence, and the fluorescence signals are evenly distributed throughout the cells (2102 in
In another embodiment, GFP DNA (green fluorescence) is packaged into T4 capsid and mCherry DNA (red fluorescence) is packaged into AAV, which are conjugated also through the Soc bridges, forming the T4(GFP)-AAV(mCherry) particles. after the T4(GFP)-AAV(mCherry) particles are added to HEK293 cells, both green and red fluorescence signals are also evenly distributed and highly merged (2104 in
In both embodiments, the correlation coefficients of green and red fluorescence signals are quantified using linear Pearson (rp) and nonlinear Spearman's rank (rs). The results show high rp and rs values for both (for T4(mCherry)-AAV(GFP): rp=0.881, rs=0.884; for T4(GFP)-AAV(mCherry): rp=0.856, rs=0.902), demonstrating high co-expression of the fluorescence genes delivered by T4 and AAV.
The T4-AAV vector is also advantageous for its ability to deliver both proteins and genes, further expanding the therapeutic potential of the hybrid nanoparticle.
In one embodiment, approximately 250 copies the 116 kDa β-galactosidase from E. coli fused to Soc (Soc-β-gal) 2204 are displayed on the T4 head 2202; ˜9 molecules of ˜6.2 kb luciferase plasmid (not shown) are packaged in T4 head 2202; GFP DNA (not shown) is packaged in AAV 2206, which attached to T4 through Hoc bridges (
In
The expression of β-galactosidase enzyme delivered by T4 is tested using X-Gal substrate. The appearance of the blue color of the cleaved X-Gal substrate indicates the successful expression of the functional β-galactosidase enzyme.
In another embodiment, the T4 capsid was displayed with either GFP or GFP fused to the nuclear localization signal (NLS), packaged with mCherry plasmid DNA and attached to AAV. These T4-AAV nanoparticles were then transduced into HEK293 cells. As shown in
Furthermore, no measurable impact on cell viability was observed following transduction by T4-AAV particles, whether Soc-bridged or Hoc-bridged, even when added to a level of 106 T4-AAV vectors per cell.
Replacing AAV with a Variant Lead to Failure in Enhancing Delivery Efficiency
In one embodiment, a phospholipase A2 (PLA2) of AAV VP1 capsid protein is mutated, producing AAV-PLA2 mutant. The T4-AAV nanoparticles are assembled by attaching the AAV-PLA2 mutant to T4 either through SBA bridges or through HBBA bridges. Luciferase DNA is packaged in T4, producing the T4(Luci)-Soc-AAV-AN and T4(Luci)-Hoc-AAV-AN hybrid vectors, and delivered to HEK293 cells.
In one embodiment, T4 is mixed with AAV without binding by biotin-avidin cross bridge. Then, luciferase DNA is packaged in T4 and delivered to HEK293 cells.
According to the above embodiments, the working ratio ranges and optimal ratios at each step of T4-AAV hybrid vector production is summarized in the table below:
In addition to high delivery efficiency, long in vivo persistence is another desired feature of viral vectors. Upon its entry into nucleus, the single stranded AAV genome replicates to produce a double-stranded AAV DNA. The double-stranded AAV DNA is thought to be converted to head-to-tail concatemers via intra- or inter-molecular recomposition at the ITRs and persist as episomes for long-term transgene expression. Studies on various AAV transduced tissues including liver and muscle have shown that the concatemers persist for a long period of time, up to 22 months15, 26. Previous studies also have shown that the ITRs significantly enhanced the in vivo persistence of T4-packaged DNAs10.
In one embodiment, the AAV inverted terminal repeats (ITRs) is engineered to flank the transgene of packaged DNAs.
In a preferred embodiment, the transgene is ˜9 ITR-Luci DNA molecules packaged in T4 head, which is attached to AAV either through Soc or Hoc cross-bridges. The resulting T4(ITRs-Luci)-Soc/Hoc-AAV(GFP) particles are then injected into mice. As shown in
In one embodiment, ˜2×1011 T4(ITRs-Luci)-Soc/Hoc-AAV(GFP) particles are injected into mice intramuscularly (i.m.). The expression of luciferase in mice is monitored by whole-body imaging and quantified by measuring photon flux for 60 days.
As shown in
In one embodiment, ˜5×1011 T4(ITRs-Luci)-Soc/Hoc-AAV(GFP) particles are injected into mice intramuscularly (i.m.). The results also confirmed that the conjugated T4-AAV facilitated more efficient delivery of T4 cargo into mice cells resulting in enhanced gene expression for a longer period when compared to T4 alone (
DNA vaccines are attractive candidates for vaccine development due to their overall safety and facile production. Formulating DNA vaccines to protect the DNA from degradation and developing efficient delivery technology play a key role in the final efficacy of DNA vaccines29. Additionally, the immune responses elicited by DNA vaccine were dramatically enhanced by a homologous protein boost30. Thus, in the present invention, T4-AAV hybrid vector, which can efficiently deliver both DNA and proteins and can also sustain the expression of the delivered genes up to 60 days, is used as a multifunctional genetic and protein (prime-boost) vaccine delivery platform.
Influenza virus is the pathogen causing flu. The hemagglutinin (HA) “stem” region of the influenza virus envelope protein is a very attractive “universal” vaccine candidate31.
In one embodiment, the present invention provides a T4-AAV delivery vector containing the HA-stem (HA4900). This DNA was packaged into T4 heads at ˜10 molecules per capsid at a packaging ratio of 30:1. As shown in
In one embodiment, mice are immunized i.m. with ˜2×1011 Hoc-bridged T4(HA4900)-AAV(VRC01) particles using a prime-boost scheme without any external adjuvant. VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. The transduction of VRC01 can also be detected in mice (data not shown), indicating the capability of T4-AAV hybrid vector to package and deliver two different genes.
In one embodiment, the mice receive a prime injection and a boost injection on Day 21. The immune response shown as IgG titer is determined on Day 14, Day 35, Day 60, Day 120 and Day 180. A single injection of T4(HA4900) elicited anti-HA IgG titers in mice (Day 14) (
Strong and durable humoral immune activities are usually characterized by germinal center response and long-lived plasma and memory B cells and are highly dependent on the help of CD4+ follicular helper T cells (Tfh)33. CXCL13 [chemokine (C-X-C motif) ligand 13] chemokine is a recognized plasma biomarker for the activities of germinal center and Tfh cells in lymphoid tissue34.
Y. pestis is the pathogen causing plague. Plague antigen F1mutV is known to elicit durable immune responses against plague.
In another embodiment, the present invention provides a method of immunizing mice using hybrid T4(HA4900)-AAV nanoparticles comprising: ˜10 molecules HA4900 DNA packaged in T4, ˜590 molecules of F1mutV displayed on the surface of T4 as a Soc fusion protein, AAV attached to T4 through Hoc-bridge. The mice are immunized following the prime-boost scheme, with a boost injection 21 days after the prime injection.
In another embodiment, the mice are immunized with F1mutV-T4(HA4900)-AAV vector using the prime-boost scheme and then challenged at the challenge dose of 295 LD50 [1 LD50=100 colony-forming units (CFU) in Balb/c mice] of the most lethal Y. pestis CO92 on Day 51.
In another embodiment, the mice are immunized with F1mutV-T4-AAV(HA4900) vector using the prime-boost scheme and then challenged at the challenge dose of 295 LD50 [1 LD50=100 colony-forming units (CFU) in Balb/c mice] of the most lethal Y. pestis CO92 on Day 51.
However, the attachment of AAV fails to lead to enhanced immune responses against HA, compared to T4 head alone (data not shown).
In the present invention, no signs of adverse or toxicity-related effects such as changes in serum alkaline phosphatase, weight loss, or temperature change were evident in any of the mice immunized with T4-AAV nanoparticles.
Having described the many embodiments of the present invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure, while illustrating many embodiments of the invention, are provided as non-limiting examples and are, therefore, not to be taken as limiting the various aspects so illustrated.
The plasmids pET-28b-Soc, pET-28b-Soc-β-gal, and pET-28b-F1mutV-Soc were constructed as previously described10, 12. For the construction of pET-28b-BAP-Hoc, two rounds of PCR were performed to amplify BAP-Hoc using three primers: FW1=ATCGAGTGGCACGAGGGTCTTTCGATGACTTTTACAGTTGATATAACTCC (SEQ ID NO: 1), FW2=TTCTAGCTAGCGGTCTTAACGACATCTTCGAGGCACAGAAGATCGAGTGGCACG AGGGTCT TCG (SEQ ID NO: 2), and RW=ATAAAGCTTTTATGGATAGGTATAGATGATACCAGTTTC (SEQ ID NO: 3). The first round of CR was performed by fusing Hoc to part of the BAP sequence using the FW1 and RW primers
Full-length BAP-Hoc was obtained by the second round of PCR using the FW2 and RW primers and then digested with NheI and HindIII. The digested BAP-Hoc fragment was sub-cloned into the ET-28b vector. The BirA-expressing plasmid pET-21a-BirA was purchased from Addgene® plasmid #20857). Plasmids pAAV-DJ, pAAV-Helper, pAAV-GFP, pAAV-luciferase, and pAAV-mCherry were purchased from Cell Biolabs™. To clone DJ-H75A-D76N mutant, upstream fragment was amplified using the primers AN-F (CGCGGAAGCTTCGATCAACTACGCAGACAG) (SEQ ID NO: 4) and HD-AN-R (GTTTGCCTCGAGGGCCGCGGCGTCTGCCTC) (SEQ ID NO: 5) and the template pAAV-DJ, and downstream fragment was amplified using the primers HD-AN-F (CCGCGGCCCTCGAGGCAAACAAAGCCTACGACCGGCAGCTCGACA) (SEQ ID NO: 6) and AN-R (GAGAACGTACGGCAGCTGGTACTCCGAGTC) (SEQ ID NO: 7). Then, the two fragments were mixed in equal amounts and used this as a template to amplify DJ-H75A-D76N using the primers AN-F and AN-R. The PCR products were subsequently cloned into the pAAV-DJ vector at the HindIII/BsiWI restriction sites to construct pAAV-DJ-H75A-D76N. Influenza HA4900 DNA fragment was synthesized by Invitrogen® according to a recent report31 and then sub-cloned into the pAAV vector after digestion with EcoRI and HindIII. All the constructed plasmids were sequenced to confirm correct fragment insertion (Retrogen®, CA).
The recombinant proteins expressed in Escherichia coli (E. coli) BL21 (DE3) RIPL cells were purified according to previously described protocols10, 12. Influenza HA4900 was purified from HEK293F suspension cells (Thermo Fisher Scientific®, MA) according to previous reports31. Briefly, the pAAV-ITRs-HA4900 plasmid was transiently transduced into HEK293F cells maintained in FreeStyle® 293 expression medium (Thermo Fisher Scientific®, MA) using the 293Fectin™ transfection reagent (Thermo Fisher Scientific®, MA). The cells were then incubated at 37° C. and 8% CO2 while shaking at 130 rpm overnight. After 12 h, an equal volume of fresh medium supplemented with sodium butyrate solution (enhancing protein expression, 2 nM final concentration) (Sigma-Aldrich®, MO) was added to the cells. On day 7, the pure supernatants were harvested by centrifugation and filtered over 0.22-μm filters (Sartorius Stedim Biotech, Germany). Proteins were purified using HisTrapHP and gel filtration columns (GE Healthcare™, IL).
The 10-amber 13-amber hoc-del soc-del T4 heads were purified according to previously described protocols6. E. coli P301 (sup-) cells (500 mL) infected with this mutant were lysed in 40 mL of Pi-Mg buffer (26 mM Na2HPO4/68 mM NaCl/22 mM KH2PO4/1 mM MgSO4, pH 7.5) containing 10 μg/mL DNaseI and chloroform (1 mL) and incubated at 37° C. for 30 min. The lysate was subjected to two low-speed (6,000×g for 10 min) and high-speed (35,000×g for 45 min) centrifugations, and the final heads pellet was resuspended in 200 μL of Tris.Mg buffer (10 mM Tris.HCl, pH 7.5/50 mM NaCl/5 mM MgCl2) and purified by CsCl density gradient centrifugation. The major head band sedimented at about ⅓ from the bottom of a 5-mL gradient was extracted and dialyzed overnight against Tris-Mg buffer. The heads were further purified by DEAE-Sepharose chromatography. The peak heads fractions were concentrated and stored at −80° C.
The rAAV-DJ was produced using the triple-plasmid transfection method (Cell Biolabs®, CA) according to the manufacturer's instructions. Briefly, plasmids, including an adenovirus helper plasmid (pHelper), a rep/cap plasmid expressing rep and cap (pAAV-DJ or pAAV-DJ-H75A-D76N), and a transgene plasmid carrying the AAV transgene cassette (AAV genome) (pAAV-GFP, pAAV-mCherry, pAAV-luciferase, pAAV-HA4900, or pAAV-VRCO1) (1:1:1), were co-transfected into HEK293 cells expressing adenovirus E1a and E1b proteins using polyethyleneimine (PEI) (Polysciences, PA). After 72 h, cells were harvested by centrifugation at 1,140×g for 10 min. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 8.5, 0.15 M NaCl) and lysed by three freeze/thaw cycles in dry ice-ethanol and a 37° C. water bath. Benzonase was added to the mixture (50 U/ml final concentration), and the lysate was incubated for 30 min at 37° C. The lysate was clarified by centrifugation at 3,700×g for 20 min, and the virus-containing supernatant was considered to be the crude lysate. Viruses were then purified by discontinuous iodixanol density gradient centrifugation at 500,000 rpm for 16 h at 4° C. (Type 55 Ti rotor, Beckman®, CA) and by HiTrap AVB Sepharose® HP according to the manufacturer's instructions (GE Healthcare™, IL). The peak fractions were dialyzed against PBS-MK buffer (PBS, pH 7.4, 2.5 mMKCl, and 1 mMMgCl2) in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific®, MA). The dialyzed AAV particles were concentrated and stored at −80° C. AAV titers were determined using a QuickTiter AAV Quantitation kit (Cell Biolabs®, CA).
For in vitro DNA packaging assays, each 20 μl reaction mixture contained purified T4 heads (˜2×1011 particles), purified full-length gp17 (˜3 μM), and linearized DNA in packaging buffer (30 mM Tris-HCl, pH 7.5, 100 mM NaCl, 3 mM MgCl2, and 1 mM ATP). The mixture was incubated at 37° C. for 30 min, followed by benzonase nuclease addition and incubation at 37° C. for 20 min to remove excess unpackaged DNA. The encapsidated nuclease-resistant DNA was released by treatment with 50 mM ethylenediaminetetraacetic acid (EDTA), 0.5 μg/μl proteinase K (Thermo Fisher Scientific®, MA), and 0.2% SDS for 30 min at 65° C. The packaged DNA was analyzed by 1% (wt/vol) agarose gel electrophoresis followed by staining with ethidium bromide, and the amount of packaged DNA was quantified using Quantity One software (Bio-Rad™, CA). The packaging efficiency was defined as the number of DNA molecules packaged per T4.
In vitro protein display on the T4 head was assessed by the co-sedimentation described previously8. Briefly, after encapsidating linearized DNA as described above, T4 heads were incubated with Soc- and/or Hoc-fusion proteins at 4° C. for 45 min. The mixtures were sedimented by centrifugation at 30,000×g for 45 min, and unbound proteins in the supernatants were removed. After washing twice with PBS, the pellets were incubated at 4° C. overnight and then resuspended in PBS for SDS/PAGE analysis or Opti-MEM for transduction. After Coomassie BlueR250 (Bio-Rad™, CA) staining and destaining, the protein bands on SDS-PAGE gels were scanned and quantified by laser densitometry (PDSI, GE Healthcare™, IL). The densities of the Hoc, Soc, and gp23 bands were determined for each lane separately, and the copy numbers of bound Hoc or Soc fusion molecules per capsid were calculated using gp23 as the internal control (930 copies per capsid).
AAV vectors and Soc proteins were biotinylated using EZ-Link™ Sulfo-NHS-LC-biotin according to the manufacturer's instructions (Thermo Fisher Scientific®, MA). Purified AAV vectors (1×1012 particles) were incubated with 500 nmol of Sulfo-NHS-LC-biotin at 37° C. for 2 h in PBS buffer. For Soc protein biotinylation, a 20-fold molar excess of the biotin reagent was used to label 10 mg of Soc in PBS buffer. To remove free biotin, the reaction mixture was dialyzed against PBS-MK buffer in Slide-A-Lyzer® dialysis cassettes (Thermo Fisher Scientific®, MA). Dialyzed bioAAV and bioSoc were concentrated and stored at −80° C.
A 15-amino acid biotin acceptor peptide (BAP, GLNDIFEAQKIEWHE) (SEQ ID NO: 8) was fused to the N-terminus of the Hoc protein. The E. coli enzyme biotin ligase (BirA) sequence specifically ligates biotin to BAP. Briefly, His6-tagged BirA and Hoc-BAP were induced in BL21RIPL cells and purified via a HisTrapHP column and SEC. For Hoc-BAP biotinylation, 1 μM recombinant BirA ligase was added to 30 μM Hoc-BAP monomer (in PBS-MgCl2 buffer, pH 7.4) in the presence of 0.3 mM biotin and 5 mM ATP. The reaction proceeded at room temperature (RT) for 3 h. Biotinylation was confirmed by streptavidin-HRP western blot analysis. Free biotin was removed by Zeba™ Desalt spin columns (Thermo Fisher Scientific®, MA). The biotinylation reaction mixture was loaded onto the center of the compact resin bed and centrifuged at 1,000×g for 2 min to collect the desalted sample. Purified Hoc-BAP-biotin was incubated with avidin at a 1:3 ratio for 1 h at 4° C. HBBA was purified by SEC. The peak fractions were concentrated and stored at −80° C.
For assembly of T4-Soc-AAV vectors on nickel beads, T4-Soc(His6)-Biotin vectors were loaded onto Ni2+-NTA agarose beads (Qiagen®, Netherlands). After incubation for 1 h at 4° C., the mixture was centrifuged at 100×g for 30 s and washed five times with binding buffer. Avidin dissolved in binding buffer was then added to the beads. After 20 min of incubation at 4° C., free avidin was removed by washing and centrifugation. Biotinylated AAV vectors were then added to the T4-Soc-SBA immobilized beads and incubated for 30 min. After washing five times with binding buffer, bound T4-Soc-AAV vectors were eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 300 mM imidazole). The protocol for assembling T4-Hoc-AAV on nickel beads was similar to that utilized for T4-Soc-AAV. Finally, the eluted vectors were exchanged into PBS-MK buffer.
AAV or T4-AAV particles were boiled in loading buffer for 10 min, separated by 12% SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad™, CA). Blocking was performed in 5% BSA/PBS-T buffer (PBS, pH 7.4, 0.05% Tween-20) at RT for 1 h with gentle shaking. Blots were then washed three times with PBS-T. Primary antibodies were added to the blots and incubated overnight at 4° C. in PBS with 5% BSA. AAV VP proteins were detected using the AAV capsid protein-specific antibody B1 (diluted 1:50, American Research Products, MA), which recognizes VP1, VP2, and VP3 based on their identical C-terminal regions. After washing with PBS-T three times, a secondary goat anti-mouse HRP-conjugated antibody (Thermo Fisher Scientific®, MA) was applied at a 1:2,000 dilution in 5% BSA/PBS-T for 1 h at RT, followed by rinsing three times with PBS-T. The bio-AAV, bio-Soc, and Hoc-BAP-biotin were directly detected by HRP-conjugated streptavidin (Abcam®, UK). Signals were visualized with an enhanced chemiluminescence substrate (Bio-Rad™, CA) using the Bio-Rad™ Gel Doc XR+ system and Image Lab software according to the manufacturer's instructions (Bio-Rad™, CA).
To label the T4 capsid with amine-reactive Alexa Fluor® 594 (Thermo Fisher Scientific®, MA), the pelleted T4 heads were resuspended in 0.1 M carbonate buffer, pH 9.0. Alexa Fluor® 594 was added to a final concentration of 0.2 mg/ml. After incubation for 1 h at room temperature with rotation in the dark, unbound dye was removed via buffer exchange into PBS, pH 7.4.
The T4-AAV complex was applied to the carbon grid for 5 min at room temperature. The grid was then frozen in liquid nitrogen using Gatan® CP3 cryo-plunger. The cryo-EM images of T4, AAV, and T4-AAVcomplex were kindly collected by Dr. Qianglin Fang at Purdue University using a Titan Krios microscope equipped with a charge-coupled device (CCD) camera. AAV particles for this experiment were provided by Dr. Mavis Agbandje-McKenna, University of Florida.
HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco®) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific®, MA), 1×HEPES (Thermo Fisher Scientific®, MA), and 1% antibiotics (Thermo Fisher Scientific®, MA) (complete DMEM). Cells were passaged with 0.05% trypsin/EDTA at a sub-cultivation ratio of 1:5 at 80-90% confluence and incubated in a humidified atmosphere at 37° C. and 5% CO2.
HEK293 cells were seeded in 24-well plates at 2.0×105 cells per well in complete DMEM. After 24 h, the cells were incubated with the AAV, bio-AAV, T4 or T4-AAV vectors at different MOIs in antibiotic-free Opti-MEM for 6 h. Thereafter, Opti-MEM was removed and replaced with complete DMEM. The cells were further incubated at 37° C. for an additional 48 h. GFP/mCherry transgene expression was observed by fluorescence microscopy (Carl Zeiss, Germany) at 48 h post-transduction, and the mean fluorescence intensities were quantified by ImageJ software. The nucleus was stained by Hoechst 33342 (Thermo Fisher Scientific®, MA). To analyze luciferase gene delivery into cells by T4 or the T4-AAV, we measured luciferase activity with the Luciferase Assay System (Promega®, WI) according to the manufacturer's instructions. Briefly, growth medium was removed, and cells were rinsed with PBS buffer. After removing the wash buffer, 150 μl of passive lysis buffer was added to each well, followed by gentle shaking at RT for 20 min. Twenty microliters of the cell lysate were then transferred to a 96-well white opaque plate and mixed with 80 μl of Luciferase Assay Reagent, and the luminescence signal was recorded by the Glomax Multi Detection System (Promega®, WI). The activity of the Soc-3-gal enzyme displayed on the T4 head in cells was determined by staining with X-Gal using the β-Galactosidase Staining kit (Sigma-Aldrich®, MO). Triplicate measurements were applied to each group.
Images were processed using Zen (Carl Zeiss, Germany) and ImageJ software. To quantify the extent of co-localization for a two-color comparison, the linear Pearson (rp) and nonlinear Spearman's rank (rs) correlation coefficients for pixels that represented the fluorescence signals in the green and red channels were calculated using the ImageJ PSC plugin.
After transfection for 48 h, cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay Kit (Promega®, WI) following the manufacturer's instructions. Briefly, an equal volume of CellTiter-Glo® Reagent was added to the cell culture in each well. The mixture was placed on an orbital shaker for 2 min to induce cell lysis and then incubated at RT for 10 min to stabilize the luminescence signal, which was recorded by the Glomax Multi Detection System (Promega®, WI). The viability of the untreated cell group was arbitrarily set to 100%, and triplicate measurements were applied to each group.
In vivo Bioluminescence Imaging was conducted as previously described10. Specifically, 5×1010 and 2×1011 T4 or T4-AAV particles were intramuscularly injected into BALB/c mice. At 0.25 day (d), 0.5 d, 1 d, 2 d, 5 d, 10 d, 15 d, 30 d, and 60 d post-administration, 30 μg of RediJect D-Luciferin Ultra (Perkin-Elmer™, MA), a luciferase substrate dissolved in 0.9% saline, was injected intraperitoneally. After 5 min, the mice were lightly anesthetized with 2% isoflurane and placed on an IVIS 200 bioluminescence whole-body imaging workstation (Caliper™). The bioluminescence emission signal was quantified using the camera control program, Living Image software, and displayed in physical units of surface radiance, photons per second per centimeter squared per steradian (photons/second/cm2/sr).
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of America (Washington, D.C.) and the University of Texas Medical Branch (Galveston, Tex.). Mice (BALB/c, female, 6-8 weeks old, Jackson Laboratories, ME) were randomly grouped and allowed to acclimate for 7 days, followed by intramuscular immunizations into their hind legs with vectors (priming on day 0 and boosting on day 21). A group of mock-immunized mice (PBS only) was included as negative control. Blood was drawn from each animal on days 0 (pre-bleed), 14, 21, 35, 60, 120, and 180, and the isolated sera were stored at −80° C.
ELISA plates (Evergreen Scientific, CA) were coated with 0.1 μg of protein per well in coating buffer (0.05 M sodium carbonate-sodium bicarbonate, pH 9.6) overnight at 4° C. After washing three times with PBS-T buffer, the plates were blocked with PBS-3% BSA buffer for 1 h at 37° C. The concentrations of antigen-specific IgG/G1/G2a in sera were monitored using a 5-fold dilution series beginning with an initial 100-fold dilution in PBS-1% BSA. The diluted serum samples were added to each well, and the plates were incubated at 37° C. for 1 h and washed five times with PBS-T buffer. The secondary goat anti-mouse IgG-HRP antibody (HRP-conjugated goat anti-mouse IgG1 or IgG2a secondary antibodies were used for IgG1/IgG2a subtypes, Invitrogen®) was then added to each well at a 1:5,000 dilution and incubated for 1 h at 37° C., followed by washing five times with PBS-T buffer. Next, the TMB (3,3′,5,5′-tetramethylbenzidine) Microwell Peroxidase Substrate System (KPL) was applied in the dark for color development. After 10 min, the enzymatic reaction was quenched by adding TMB BlueSTOP (KPL) solution, and plates were read within 30 min at 650 nm by an ELISA reader (VERSA max, Molecular Devices). Endpoint titers were presented as the sample dilution resulting in an OD650 equal to twice the mean background (negative serum) of the assay. For CXCL13 quantification in sera, the mouse CXCL13 ELISA kit (Boster Biological Technology, CA) was used by following the manufacturer's instructions.
The ALP biochemical parameter was determined in sera of mice immunized with T4, AAV, or T4-AAV. Naive mice were used as control. ALP level was measured using a commercial ALP assay kit (Elabscience®, MD) following the manufacturer's instructions.
The acclimated mice were intramuscularly immunized into their hind legs with T4, AAV, F1mutV-T4(HA4900), or F1mutV-T4(HA4900)-AAV (priming on day 0 and boosting on day 21). Sera were collected from each animal on days 0 (pre-bleeds) and 35 for immunological analyses. On day 42, mice were intranasally challenged with 295 LD50 of Y. pestis CO92 bacteria40. Animals were monitored and recorded for mortality, body weight, and body temperature.
All quantified data are presented as the mean±standard deviation (SD). Statistical analyses were performed by two-tailed Student's t-tests. Significant differences between two groups are indicated by *p<0.05 or **p<0.01.
A therapeutically effective amount of the hybrid vector is used as DNA and protein delivery tool or for the vaccination against infectious diseases such as, but not limited to, COVID, flu, HIV, anthrax and plague.
A therapeutically effective amount of the hybrid vector carrying DNA molecules or proteins can be administered by a variety of routes. In effecting vaccination of a subject to prevent infectious diseases such as, but not limited to, COVID, flu, HIV, anthrax and plague, the hybrid vector can be administered in any form or mode that makes the hybrid vector bioavailable in an effective amount. For example, the compounds can be administered orally, by inhalation, or by the subcutaneous, intramuscular, intravenous, transdermal, intranasal, rectal, ocular, topical, sublingual, buccal, or other routes.
One skilled in the art of preparing hybrid vector can readily select the proper form and mode of administration depending upon the particular characteristics of the hybrid vector selected, the disorder or condition to be treated, the stage of the disorder or condition, and other relevant circumstances.
The pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art, wherein the pharmaceutical compositions comprise therapeutically effective amount of compositions of the hybrid vector carrying one or more DNA molecules and proteins. The carrier or excipient may be a solid, semi-solid, or liquid material that can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art.
The pharmaceutical composition may be adapted for oral or parenteral and may be administered to the subject in the dosage form of tablets, sugar-coated tablets, capsules, delayed-release hard capsules, softgel, chewable tablets, gummy, injection fluids, caplets, powders, granules, syrups, aerosols, inhalants, suppositories, solutions, suspensions, catheters containing the composition, syringes containing the composition, implants containing the composition, transdermal patch, or the like.
In one example, a composition disclosed herein may be formulated in a dosage form of a softgel as shown in
In another example, a composition disclosed herein may be formulated in a dosage form of a tablet as shown in
In addition to above examples of dosage forms for oral administration, the disclosed composition may also be delivered to a body of a subject such as via injection. A composition may be formulated in a dosage form of an injection fluid and be loaded into an injectable device (e.g., a syringe), to inject into a subject's body.
In some examples, a composition disclosed herein may be delivered into a subject's body such as via a transdermal patch.
In some examples, as shown in
The composition may be used at appropriate dosages defined by routine testing to obtain optimal pharmacological effect, while minimizing any potential toxic or otherwise unwanted effects.
An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining an effective amount, the dose of a hybrid vector, a number of factors are considered by the attending diagnostician, including, but not limited to: the vector to be administered; the species-its size, age, and general health; the specific disorder involved; the degree of involvement or the severity of the disorder; the response of the individual subject; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of other concomitant medication; and other relevant circumstances.
The specific dose administered may be determined by particular circumstances surrounding each situation. These circumstances can include: the route of administration, the prior medical history of the recipient, the symptom being treated, the severity of the symptom being treated, and the age of the recipient. The recipient subject's attending physician should determine the therapeutic dose administered in light of the relevant circumstances.
Also, it is to be understood that the exact dose may be determined, in accordance with the standard practice in the medical arts of “dose titrating” the recipient; that is, initially administering a low dose of the compound, and gradually increasing the dose until the desired therapeutic effect is observed.
It is to be further understood that the dosage regimen can be selected in accordance with a variety of factors. These include type, species, age, weight, sex, diet, and medical condition of the subject; the severity of the condition to be treated; the route of administration; the kidney and liver functions of the subject; the time of administration; the rate of excretion; and the particular hybrid vector employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the disease or disorder that is being treated.
In one example, this product may be used for administration according to a continuous schedule having a dosing interval selected from one or more of: once daily dosing and/or multiple daily dosing. In one embodiment, this product may be administered to a subject having a need thereof chronically.
It is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.
All documents, patents, journal articles and other materials cited in the present application are incorporated herein by reference.
The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.
The following references are referred to above and are incorporated herein by reference:
All documents, patents, journal articles and other materials cited in the present application are incorporated herein by reference.
While the present invention has been disclosed with references to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the sphere and scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention is not limited to the described embodiments, but that it has the full scope defined by the language of the following claims, and equivalents thereof.
This application claims benefit of priority of U.S. Patent Application No. 62/888,576 filed on Aug. 19, 2019, entitled “A PROKARYOTIC-EUKARYOTIC HYBRID VIRAL VECTOR FOR DELIVERY OF LARGE CARGOS OF GENES AND PROTEINS INTO HUMAN CELLS”. The entire contents and disclosures of this patent application are incorporated herein by reference in their entirety.
This invention was made with the United States government support under Grant Nos. AI111538 and AI081726 awarded by NIAID AND NIH. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62888576 | Aug 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16990289 | Aug 2020 | US |
| Child | 17488542 | US |
