Current methods of utilizing chimeric antigen receptor cells to treat cancer have numerous limitations, including multiple and prolonged steps, a need for the identification of highly specific antigens, undesired side effects (e.g., side effects associated with off-target effects), and the need for the utilization of adjuvants. Numerous embodiments of the present disclosure address the aforementioned limitations.
In some embodiments, the present disclosure pertains to modified antigen presenting cells that include a recombinant protein appended onto a surface of the antigen presenting cells. In some embodiments, the recombinant protein includes an ectodomain that is positioned on the surface of the antigen presenting cells and capable of preferentially binding to an antigen of cancer cells. The recombinant protein also includes a transmembrane domain that includes at least one region embedded in the antigen presenting cell membrane that serves as an anchor for maintaining the ectodomain of the recombinant protein on the surface of the antigen presenting cells.
In additional embodiments, the recombinant protein also includes an antigen presenting cell-recruiting domain that directs the antigen presenting cells towards the cancer cells. In further embodiments, the recombinant protein also includes an antigen presenting cell activator that activates or licenses the antigen presenting cells.
In additional embodiments, the present disclosure pertains to methods of expressing the recombinant proteins of the present disclosure on antigen presenting cells by introducing a carrier into the antigen presenting cells. The carrier includes a nucleotide sequence. The recombinant protein is expressed by the antigen presenting cells from the nucleotide sequence and appended onto a surface of the antigen presenting cells.
The methods of the present disclosure can have numerous in vitro and in vivo embodiments for various purposes. For instance, in some embodiments, the methods of the present disclosure can include administering the carriers of the present disclosure into a subject to result in the preferential uptake of the carriers by antigen presenting cells and the expression of the recombinant proteins of the present disclosure on the surfaces of the antigen presenting cells. Thereafter, the modified antigen presenting cells can bind to cancer cells through an interaction between the recombinant proteins and cancer cells, thereby activating an anti-cancer immune response against the cancer cells.
In alternative embodiments, the methods of the present disclosure can include a step of introducing the carriers of the present disclosure into antigen presenting cells to result in the expression of the recombinant proteins of the present disclosure on the surfaces of the antigen presenting cells. Thereafter, the modified antigen presenting cells are introduced to a subject in order to activate an anti-cancer immune response against the cancer cells in the subject.
The methods of the present disclosure can have numerous applications. For instance, in some embodiments, the methods of the present disclosure can be utilized to treat a cancer in a subject by introducing the carriers or modified antigen presenting cells of the present disclosure to the subject. The methods of the present disclosure can be utilized to treat numerous types of cancers in subjects. For instance, in some embodiments, the cancer includes cancers associated with epithelial derived solid tumors, bone marrow-derived tumors, and combinations thereof.
Additional embodiments of the present disclosure pertain to the carriers of the present disclosure. In some embodiments, the carriers of the present disclosure are in the form of particles that encapsulate nucleotides that express the recombinant proteins of the present disclosure.
It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and/or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.
The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.
Chimeric antigen receptor (CAR) T cell therapy is an effective immunotherapy for some patients with certain kinds of cancers. By expressing an engineered receptor, T-cells could recognize cancer cells (through particular antigens on their surfaces), activate themselves (in a major histocompatibility complex (MHC) independent manner), proliferate, and motivate other components in the immune system (through the secretion of cytokines) to fine-tune the immune response against cancer cells.
The aforementioned effects are generally achieved through different domains of engineered receptors. For instance, the ectodomain of CAR is usually a single-chain variable fragment (scFv) that binds to a particular antigen, such as CD19 on B cell malignancies, which define the specificity and safety of CAR-T cell therapy.
CD3 zeta chain, the signal-transduction component of the T cell receptor (TCR)-CD3 complex, is the most important endodomain as it transmits an activation signal to the T cell after antigen-scFv binding. Other co-stimulatory domains, such as CD28 and 4-1BB, are effective in transmitting proliferative or survival signals to fully activate T-cells.
However, there are still many challenges to overcome for the applications of CAR-T in a broad range of cancer types, such as solid tumors. For instance, the heterogeneity of cancer cells makes it difficult to identify suitable antigens. Moreover, the non-specific antigen may compromise the safety and efficiency of the treatment. Furthermore, the efficacy of CAR-T is limited by the immunosuppressive cancer microenvironment, which is comprised of multiple immunosuppressive cell types, cytokines and suboptimal conditions such as hypoxia and acidification.
Many efforts have been made to reinforce the functions of CAR-T in the cancer microenvironments. Such efforts have included the design of more efficient CAR-T and their therapeutic combination with small molecules. However, only limited effectiveness has been observed.
Additionally, CAR-T treatment is time and effort consuming, making it too expensive for the majority of patients. Generally, T cells must first be collected from patients, genetically modified to express CAR, expanded for several weeks to obtain enough functional CAR-T cells, and then infused back to patients under lympho-depleting chemotherapy. These complicated procedures call for dedicated equipment and expertise, thereby making them difficult to apply on a large scale.
Additional limitations exist for the use of CAR-T. For instance, to obtain better therapeutic effects, many CAR-T therapies require the need for the utilization of adjuvants to activate the antigen presenting cells. Additionally, numerous side effects are associated with the administration of the chimeric antigen receptor cells and adjuvants to subjects.
As such, a need exists for modified antigen presenting cells that can be used to treat numerous types of cancers in various subjects in a cost effective and efficient manner. Numerous embodiments of the present disclosure address the aforementioned need.
In some embodiments, the present disclosure pertains to modified antigen presenting cells that include a recombinant protein appended onto a surface of the antigen presenting cells. In some embodiments, the recombinant protein includes an ectodomain that is positioned on the surface of the antigen presenting cells and capable of preferentially binding to an antigen of cancer cells. The recombinant protein also includes a transmembrane domain that includes at least one region embedded in the antigen presenting cell membrane that serves as an anchor for maintaining the ectodomain of the recombinant protein on the surface of the antigen presenting cells.
In additional embodiments, the recombinant protein also includes an antigen presenting cell recruiting domain that directs the antigen presenting cells towards the cancer cells. In further embodiments, the recombinant protein also includes an antigen presenting cell activator that activates or licenses the antigen presenting cells.
In some embodiments illustrated in
In additional embodiments, the present disclosure pertains to methods of expressing the recombinant proteins of the present disclosure on antigen presenting cells by introducing a carrier into the antigen presenting cells. The carrier includes a nucleotide sequence. The recombinant protein is expressed by the antigen presenting cells from the nucleotide sequence and appended onto a surface of the antigen presenting cells.
The methods of the present disclosure can have numerous in vitro and in vivo embodiments for various purposes. For instance, as illustrated in
Likewise, as illustrated in
As further illustrated in
Additional embodiments of the present disclosure pertain to the carriers of the present disclosure. In some embodiments, the carriers of the present disclosure are in the form of particles that encapsulate nucleotides that express the recombinant proteins of the present disclosure. In some embodiments illustrated in
As set forth in more detail herein, the carriers, modified antigen presenting cells and methods of the present disclosure can have numerous embodiments. In particular, numerous types of recombinant proteins may be appended onto surfaces of numerous antigen presenting cells for interaction with various types of cancers in various subjects through various mechanisms.
Antigen Presenting Cells
The recombinant proteins of the present disclosure may be appended to numerous antigen presenting cells. For instance, in some embodiments, the antigen presenting cells include, without limitation, macrophages, B-cells, dendritic cells, and combinations thereof.
In some embodiments, the antigen present cells include dendritic cells. In some embodiments, the use of dendritic cells (DCs) as modified antigen presenting cells are advantageous because they link innate to adaptive immunity, thereby enabling the generation of anti-tumor immune responses against a variety of cancer cell antigens, despite of their heterogeneity.
For instance, in some embodiments, immature dendritic cells have a strong ability to uptake and present antigens, such as debris from cancer cells, and become mature under adverse signals in a tumor microenvironment. These mature dendritic cells can then home to lymph nodes and become licensed by antigen specific CD4+ T cells through CD40-CD40L interactions. Thereafter, licensed dendritic cells can activate cognate CD8+ T cells and trigger tumor regression or eradication.
Recombinant Proteins
In general, the recombinant proteins of the present disclosure include an ectodomain and a transmembrane domain. In additional embodiments, the recombinant proteins of the present disclosure also include an antigen presenting cell recruiting domain. In further embodiments, the recombinant proteins of the present disclosure also include an antigen presenting cell activator. As set forth in more detail herein, the recombinant proteins of the present disclosure include various ectodomains, transmembrane domains, recruiting domains, and antigen presenting cell activators.
Ectodomains
An ectodomain generally refers to a region of a recombinant protein that is positioned on the surface of the antigen presenting cells of the present disclosure. In addition, the ectodomain is capable of preferentially binding to an antigen of cancer cells.
The recombinant proteins of the present disclosure can include numerous ectodomains. For instance, in some embodiments, the ectodomains of the present disclosure include, without limitation, antibodies, single chain antibodies, nanobodies, aptamers, antibody fragments, portions of antibodies, scFV portions of antibodies, peptides, and combinations thereof. In some embodiments, the ectodomains of the present disclosure include an antibody fragment.
The ectodomains of the present disclosure can preferentially bind to numerous antigens of cancer cells. For instance, in some embodiments, the cancer cell antigen includes, without limitation, moieties, proteins, glycoproteins, EGF receptors, MUC-1, lipids, EPCAM, HER-2 receptors, and combinations thereof.
In some embodiments, the cancer cell antigens include an EPCAM protein, and the ectodomain is an anti-EPCAM antibody. In some embodiments, the cancer cell antigen is a HER-2 receptor, and the ectodomain is an anti-HER-2 antibody.
Transmembrane Domains
A transmembrane domain generally refers to a recombinant protein site that includes one region that is embedded in the antigen presenting cell membrane. The transmembrane domain also serves as an anchor for maintaining the ectodomain on the surface of the antigen presenting cells.
The recombinant proteins of the present disclosure can include numerous transmembrane domains. For instance, in some embodiments, the transmembrane domain includes, without limitation, glycosylphosphatidylinositol (GPI)-anchored domains, glycolipid-linked domains, CD8-based transmembrane domains, CD4-based transmembrane domains, TLR-based transmembrane domains, and combinations thereof.
In some embodiments, the transmembrane domain includes a glycosylphosphatidylinositol (GPI)-anchored domain. In some embodiments, the GPI-anchored domain can be from numerous GPI-anchored proteins. For instance, in some embodiments, the proteins include, without limitation, CD73, CD58, CD59, DAF, CD14, and combinations thereof.
Recruiting Domains
A recruiting domain generally refers to a recombinant protein site that directs the antigen presenting cells towards cancer cells. In some embodiments, the recruiting domain is positioned on the recombinant protein ectodomain.
The recombinant proteins of the present disclosure can include numerous types of recruiting domains. For instance, in some embodiments, the recruiting domains include a receptor. In some embodiments, the receptor directs the antigen presenting cells to cancer cells that secrete a protein that binds to the receptor. In some embodiments, the receptor is CCR6, and the secreted protein is CCL20. In some embodiments, the receptor is an IL-10 receptor, and the secreted protein is IL-10.
Antigen Presenting Cell Activators
Antigen presenting cell activators generally refer to recombinant protein sites that activate or license antigen presenting cells. In some embodiments, the antigen presenting cell activators are positioned on a recombinant protein endodomain.
The antigen present cell activators of the present disclosure can activate antigen presenting cells through numerous mechanisms. For instance, in some embodiments, the antigen presenting cell activators activate the antigen presenting cells through upregulation of the expression of proteins. In some embodiments, the upregulated proteins include, without limitation, major histocompatibility complex proteins, co-stimulatory proteins, pro-inflammatory cytokines, toll-like receptors, or combinations thereof.
The antigen present cell activators of the present disclosure can license antigen presenting cells through numerous mechanisms. For instance, in some embodiments, the antigen presenting cell activators of the present disclosure license antigen presenting cells through upregulation of proteins to efficiently induce a CD8+ T cell response. In some embodiments, the upregulated proteins include, without limitation, OX40L, 4-1BBL, and combinations thereof.
The recombinant proteins of the present disclosure can include numerous antigen presenting cell activators. For instance, in some embodiments, the antigen presenting cell activators include, without limitation, CD40, TLR4, TLR2, TLR3, TLR7, TLR8, TLR9, TLR5, FLT3, 4-1BB, LTBR, RANK, and combinations thereof. In some embodiments, the antigen presenting cell activator includes TLR4 and CD40.
In some embodiments, the antigen presenting cell activator includes CD40. In some embodiments, the antigen presenting cells are activated after signal transduction by CD40 to upregulate expression of MHC II, CD80, co-stimulatory protein CD86, pro-inflammatory cytokines, or combinations thereof.
In some embodiments, the antigen presenting cell activator is TLR4. In some embodiments, the antigen presenting cells are activated after signal transduction (e.g., signal transduction by TLR4) to upregulate expression of MHC II, CD80, co-stimulatory protein CD86, pro-inflammatory cytokines, or combinations thereof.
Expression of Recombinant Proteins on Antigen Presenting Cells
Various methods may be utilized to express the recombinant proteins of the present disclosure on antigen presenting cells. For instance, in some embodiments, the expression occurs by introducing a carrier of the present disclosure into the antigen presenting cells. Thereafter, the recombinant protein is expressed by the antigen presenting cells from the nucleotide sequence of the carrier and appended onto a surface of the antigen presenting cells.
The recombinant proteins of the present disclosure can be expressed on antigen presenting cells in various manners. For instance, in some embodiments, the carriers of the present disclosure are introduced into antigen presenting cells in vitro. In some embodiments, the introduction results in the expression of the recombinant proteins of the present disclosure on antigen presenting cells in vitro. Thereafter, the modified antigen presenting cells can be introduced into a subject.
In some embodiments, the carriers of the present disclosure are directly introduced into a subject. In some embodiments, the introduction results in the preferential uptake of the carriers by antigen presenting cells of the subject. In some embodiments, the preferential uptake results in the expression of the recombinant proteins of the present disclosure on antigen presenting cells in vivo.
Various methods may be utilized to introduce the carriers and modified antigen presenting cells of the present disclosure to subjects. For instance, in some embodiments, the introduction occurs by an administration method that includes, without limitation, intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, oral administration, intratumor administration, and combinations thereof. In some embodiments, the administering occurs by intravenous administration.
Subjects
The recombinant proteins of the present disclosure may be expressed by antigen presenting cells in various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is suffering from cancer. As such, in some embodiments, the methods of the present disclosure can be utilized to treat the cancer in the subject.
Treatment of Cancers
The methods of the present disclosure can be utilized to treat numerous types of cancers in subjects. For instance, in some embodiments, the cancer includes cancers associated with epithelial derived solid tumors, bone marrow-derived tumors, and combinations thereof.
In some embodiments, the cancers to be treated include cancers associated with epithelial derived solid tumors. In some embodiments, such cancers include, without limitation, breast cancer, gastrointestinal carcinomas, head and neck cancer, hepatocellular carcinoma, lung cancer, ovarian cancer, pancreatic cancer, and combinations thereof.
In some embodiments, the cancers to be treated include cancers associated with bone marrow-derived tumors. In some embodiments, such cancers include, without limitation, leukemia, multiple myeloma, and combinations thereof.
The modified antigen presenting cells of the present disclosure can be utilized to treat cancers in subjects through various mechanisms. For instance, in some embodiments, the modified antigen presenting cells of the present disclosure treat cancers in subjects by enabling the immune system to attack the cancer cells of the subject.
In some embodiments, the modified antigen presenting cells of the present disclosure enable T-cells to attack cancer cells by binding to the cancer cells through interaction between the recombinant protein ectodomain of the antigen presenting cells and the antigen of the cancer cells. Thereafter, the interaction results in the presentation of the antigen of the cancer cells on a surface of the antigen presenting cells. Next, the antigen presenting cells present the antigen of the cancer cells on the surface of the antigen presenting cells to the T-cells. As a result, the T-cells become activated to initiate anti-cancer immune responses against the cancer cells.
In some embodiments, the modified antigen presenting cells that activate T-cells include dendritic cells. In some embodiments, the modified antigen presenting cells that activate T-cells include macrophages. In some embodiments, the macrophages bind to the cancer cells through interaction between the recombinant protein ectodomain of the macrophages and the antigen of the cancer cells to result in the killing of the cancer cells by the macrophages. In some embodiments, the killing of the cancer cells by the macrophages occurs by phagocytosis of the cancer cells by the macrophages.
Carriers
Carriers generally refer to delivery vectors that are able to express the recombinant proteins of the present disclosure in antigen presenting cells and thereby form the modified antigen presenting cells of the present disclosure. The carriers of the present disclosure generally include a nucleotide sequence that expresses the recombinant proteins of the present disclosure.
The carriers of the present disclosure can be in various forms. For instance, in some embodiments, the carriers of the present disclosure include, without limitation, particles, microparticles, nanoparticles, micelles, lentiviruses, retroviruses, and combinations thereof. In some embodiments, the carriers of the present disclosure are in the form of nanoparticles.
The nucleotide sequences in the carriers of the present disclosure can also be in various forms. For instance, in some embodiments, the nucleotide sequence includes, without limitation, DNA, mRNA, and combinations thereof. In some embodiments, the nucleotide sequence includes a DNA on a plasmid.
In some embodiments, the nucleotide sequences of the present disclosure may be encapsulated within the carriers of the present disclosure. For instance, in some embodiments, the nucleotide sequences of the present disclosure may be encapsulated within particles. In some embodiments, the nucleotide sequences may also be embedded with polymers.
Applications and Advantages
The methods and modified antigen presenting cells of the present disclosure provide numerous advantages. For instance, in some embodiments, the modified antigen presenting cells of the present disclosure are expected to provide minimal side effects when introduced to subjects. For instance, in some embodiments where there is no mutated gene in normal tissue to trigger specific immune responses, the off-target side effects are negligible (i.e., binding of modified antigen presenting cells to normal cells will not trigger a specific immune response to kill normal cells). Moreover, since the modified antigen presenting cells of the present disclosure can be activated through interaction with cancer cells, no additional adjuvants are necessary to provide such activation.
Furthermore, in some embodiments, the methods and carriers of the present disclosure can be utilized to preferentially express the recombinant proteins of the present disclosure on the antigen presenting cells of subjects in vivo, thereby eliminating the need for harvesting and modifying the antigen presenting cells in vitro. As such, the modified antigen presenting cells and methods of the present disclosure can be used to treat numerous types of cancers in various subjects in a cost effective and efficient manner.
Moreover, in some embodiments, after binding to cancer cells, the modified antigen presenting cells of the present disclosure (e.g., GPI anchored scFv modified DCs or CAR DCs) could uptake antigens from cancer cells to readily trigger epitope spreading. Therefore, in such embodiments, there is no need to identify highly specific antigens for each patient. A general antigen to guide the modified antigen presenting cells of the present disclosure (e.g., GPI anchored scFv modified DCs or CAR DCs) to cancer sites to start this process can be sufficient. For instance, in some embodiments, the EPCAM protein on epithelial derived solid tumors is a sufficient target.
Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.
This Example demonstrates the expression of a single chain variable fragment (scFv) on dendritic cell (DC) membranes either through GPI anchors (modified cells referred to herein as “GPI anchored scFv modified DC”) or chimeric antigen receptors (modified cells referred to herein as “CAR DC”). The dendritic cells efficiently target and bind to cancer cells. Thereafter, the dendritic cells get fully activated through the binding. The fully activated dendritic cells can uptake and present cancer specific antigens, express co-stimulatory molecules and produce pro-inflammatory cytokines to activate the immune system.
Numerous differences exist between CAR DC and GPI anchored scFv modified DC. For instance, there are some intracellular domains in CAR DC structures that are absent from GPI anchored scFv modified DC. After binding with cancer cells, these intracellular domains could fully activate dendritic cells. On the other hand, in order to activate GPI anchored scFv modified DC, the cells must be treated with adjuvants, such as LPS or CpG. 2.
Moreover, strategies used to translocate fusion proteins to cell membranes are different in CAR DC and GPI anchored scFv modified DC. For instance, CD8 transmembrane domains are utilized for CAR DC while a GPI anchor is used for GPI anchored scFv modified DC.
To translocate the scFv to the surface of DC, Applicant fused scFv with an N-terminus secretion signal and a c-terminus Glycosylphosphatidylinositol (GPI) anchor. These two domains help the fusion protein to be recognized and sorted to the endoplasmic reticulum (ER) and finally anchor to the cell membrane through a secretory pathway.
Dendritic cells expressing this kind of GPI anchored scFv could bind with particular cancer cells, uptake antigen from them, and then activate specific immune responses against them. As such, the aforementioned dendritic cells provide an optimal platform and could be utilized to apply numerous scFvs to recognize numerous molecules on cancer cell surfaces. In order to establish preliminary data for the aforementioned platform, Applicant utilized anti-EpCAM GPI (aEpCAM-GPI) or anti-Her2 GPI (aHer2-GPI).
In the example illustrated in
For the intracellular domain, Applicant utilized TLR4 and CD40 intracellular domains in tandem. TLR4 is the receptor of LPS (lipopolysaccharide), the most efficient dendritic cell agonist. CD40 signaling is desirable for dendritic cell licensing. Applicant believes that the binding of dendritic cells to cancer cells through aEpCAM scFv could activate TLR4 and CD40 signaling pathways, which could fully activate and license dendritic cells, thereby making them ready to activate the immune system.
In the N-terminal, Applicant added a Myc tag to detect the expression of CAR. In addition, the 5′UTR and 3′UTR+poly A were utilized to enhance the expression efficacy of CAR.
For the experiments in
To further affirm that the CAR TLR4 and CD40 intracellular domains can activate dendritic cells, Western blot analyses were performed on the cells after CAR expression to test for activation of downstream signaling pathways. The results, which are summarized in
In order to fully activate an immune response, dendritic cells should preferably secrete many cytokines, including TNFa and IL-6. To further affirm the activation of dendritic cells, Applicant co-cultured normal BMDC, BMDC expressing EGFP-GPI or CAR with cancer cells (B16 expressing hEpCAM) and measured TNFa and IL-6 expression. The results, which are summarized in
In order to test the therapeutic efficacy of aEpCAM-GPI and CAR DC in vivo, Applicant used aEpCAM-GPI or CAR BMDC to treat a B16-OVA-hEpCAM tumor model. The results of this assay are shown in
In Example 1, Applicant demonstrated that dendritic cells with GPI-anchored anti-EpCAM single chain variable fragments (scFv) had anti-tumor effects. In this Example, Applicant demonstrates that that dendritic cells with GPI-anchored anti-Her2 single chain variable fragments (scFv) also have anti-tumor effects.
In this Example, Applicant provides data related to plasmid DNA (pDNA) delivery nanoparticles for delivering plasmids expressing aEpCAM-GPI or aEpCAM CAR fusion genes to dendritic cells both in vitro and in vivo. Also provided are measurements related to sizes, zeta potentials, encapsulating capacities and expression efficacies of the naoparticles in the HEK293FT cell line and the dendritic cell line DC2.4.
For the core, Applicant utilized a PBAE (poly(beta-amino ester)) polymer conjugated with MTAS-NLS (microtubule-associated sequences (MTAS) and nuclear localization signals (NLS)), a peptide guiding pDNA to the nucleus of target cells. This positive charged complex integrates with negative charged plasmids to form a condensed core.
For the shell structure, Applicant has three different combinations as listed in
The core only (the PBAE-MTAS-NLS core) provided a modest gene expression. However, the core and shell (DOPC PBAE) triggered an intensive gene expression. The results were confirmed in both HEK293FT cells and DC2.4 cells. The size of core and shell particle is around 600 nm, and the zeta potential is around 15 mV. In sum, the results indicate that both the core and shell are necessary for intensive gene expression.
Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.
This application claims priority to U.S. Provisional Patent Application No. 62/833,110, filed on Apr. 12, 2019. The entirety of the aforementioned application is incorporated herein by reference.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US20/27968 | 4/13/2020 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62833110 | Apr 2019 | US |