Targeted Non-Charged-Nucleic Acid (NCNA) Delivery and Related Tumor Penetrating Nanocomplexes

Field

[0001] This invention is in the field of targeted nucleic acid delivery for use to treat various human diseases.

Background

[0002] For the treatment of cancers, issues related to delivery can limit anticancer applications. For example, the inability to selectively target delivery to tumors and/or to penetrate tumor stroma limit cancer treatment. In addition, sequestration of some cancer drugs in in endosomes limits availability of those drugs to effect target. For example, 95% of antisense and siRNA drugs are sequestered in endosomes, adversely limiting their anticancer effect. When attempting to drive effective tumor concentrations, toxi cities associated with high doses are known to emerge. These toxicities manifest in form of clotting factors, complement and immune activation; renal toxicities; and/or another dose-dependent on-and off-target toxicities.

[0003] Accordingly, there is a need for additional compositions that can address the issues related to nucleic acid delivery to tumors for treating diseases, including cancer.

Summary

[0004] Provided herein are heterologous non-charged-nucleic acid (NCNA) constructs, tumor-penetrating-nanocomplex (TPNs); and related methods of making and using same for treating a variety of diseases. In a particular embodiment, provided herein is a heterologous non-charged-nucleic acid (NCNA) construct, comprising a targeting peptide complex; a non- charged-nucleic acid (NCNA); and an endosome escape moiety (EEM). wherein the heterologous NCNA construct is capable of forming a tumor-penetrating-nanocomplex (TPN). [0005] The invention NCNA constructs and TPNs provided herein address issues related to nucleic acid delivery (e.g., NCNA delivery) of drugs to tumors (e.g., solid tumors); and provides several advantages, such as a targeted approach to increase tumor vascular permeability. For example, the invention tumor penetrating complex that are formed by invention NCNA constructs provides tumor and/or immune cell targeting. In addition, in accordance with the methods and compositions provided herein, the CendR pathway is targeted for activation to penetrate the stroma and to deliver drug to all layers of a tumor to enable efficacious drug concentrations within solid tumors. In addition, the invention NCNA constructs and TPNs produced therefrom provide the technology to evade problematic endosome sequestration. Another advantage of the invention compositions and methods is that the targeted tissue penetration permits dose- and toxicity-sparing potency. Another advantage provide by the invention NCNA constructs and TPNs produce therefrom is their ease of synthesis compared to biologies, such as virus-like particles, antibody-conjugates or exosomes. The invention NCNA constructs and TPNs can incorporate a variety of carriers, such as LNPs and silicon core fusogenic lipid nanoparticles.

[0006] In particular embodiments, the invention TPNs can be co-administered with suitable peptides, such as LSTA-1 or iRGD, and the like.

[0007] Also provided herein are particular embodiments set forth as numbered Aspects, such as for example as Aspect 1, correspondint to a heterologous non-charged-nucleic acid (NCNA) conjugate capable of forming a tumor- penetrating-nanocomplex (TPN), comprising targeting peptide complex; a non-charged-nucleic acid (NCNA); and an endosome escape moiety (EEM), wherein the heterologous NCNA construct is capable of forming a tumor-penetrating- nanocomplex (TPN).

Aspect 2. The conjugate of Aspect 1, wherein the targeting peptide complex is a monoantennary or multi antennary targeting peptide complex.

Aspect 3. The conjugate of Aspect 2, wherein a number of targeting peptides in the multi antennary targeting peptide complex is selected from the group of ranges consisting of: 2-500, 2-450, 2-400, 2-350, 2-300, 2-250, 2-200, 2-150, 2-125, 2- 100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20 2-18, 2-16, 2-14, 2-12, 2-10, 2- 9, 2-8, 2-8, 2-6, 2-5, 2-4, 2-3 peptides. Aspect 4. The conjugate of Aspect 3, wherein a number of targeting peptides in the targeting peptide complex is monoantennary, biantennary, triantennery and/or quadrantennary.

Aspect s. The conjugate of Aspect 1, wherein the non-charged-nucleic acid (NCNA) is covalently attached to one or both of the targeting peptide complex and/or the endosome escape moiety by a linker.

Aspect 6. The conjugate of Aspects 1-5, wherein the linker is either cleavable or uncleavable; and wherein the NCNA is between the targeting peptide complex and the EEM, or the EEM is between the targeting peptide complex and the NCNA.

Aspect 7. The conjugate of Aspects 1-6, wherein the non-charged-nucleic acid is selected from a peptide-nucleic acid (PNA), phosphorodiamidate morpholino oligonucleotide (PMO) and/or a short interfering ribonucleic neutral (siRNN).

Aspect 8. The conjugate of Aspects 1-7, wherein the endosome escape moiety is selected from one or more of: transportan, TAT peptide (RKKRRQRRR), poly-Y, and/or 6His-CM18-PTD4, a chimera of the 6His tag, PTD4 (YARAAAARQARA), and CM18 (KWKLFKKIGAVLKVLTTG).

Aspect 9. The conjugate of Aspects 1-8, wherein the targeting peptide complex comprises an iRGD peptide or LSTA1 corresponding to CAS Registry No: 2580154-02-3.

Aspect 10. The conjugate of Aspects 1-9, wherein the fatty acid moiety covalently attached to the endosome escape moiety.

Aspect 11. The conjugate of Aspects 1-10, wherein the fatty acid moiety is at the

N-terminus of the conjugate. Aspect 12. The conjugate of Aspects 1-11, further comprising a PEG covalently attached between the NCNA and targeting peptide complex and/or the NCNA and endosome escape moiety.

Aspect 13. A tumor-penetrating-nanocomplex (TPN), comprising the heterologous non-charged-nucleic acid conjugate of Aspects 1-12.

Aspect 14. The TPN of Aspect 13, wherein the TPN comprises a range of heterologous NCNA conjugates selected The TPN of Aspect 13, wherein the TPN comprises a range of heterologous NCNA conjugates selected from: 2-10 ⁶ , 2-10 ⁵ , 2-10 ⁴ , 2-1000, 2-900, 2-800, 2-700, 2-600, 2-500, 2-450, 2-400, 2-350, 2-300, 2- 250, 2-200, 2-150, 2-125, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50 copies of the heterologous NCNA conjugate.

Aspect 15. The TPN of Aspect 13, wherein the TPN comprises an amount of heterologous NCNA conjugates selected from greater than: 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2-10 ⁴ , 2-10 ⁵ , 2-10 ⁶ .

Aspect 16. A targeted non-charged-nucleic acid (NCNA) construct, comprising: targeting peptide complex; and a non-charged-nucleic acid moiety (NCNA), and optionally, an endosome escape moiety (EEM).

Aspect 17. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 16, wherein the targeting peptide complex and the NCNA are connected via a linker.

Aspect 18. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 16-17, wherein the linker is non-cleavable. Aspect 19. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 16, wherein the targeting peptide complex and the NCNA moiety are connected as a single, contiguous polypeptide.

Aspect 20. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 16-19, further comprising a PEG moiety.

Aspect 21. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 20, wherein the PEG moiety is between the targeting peptide complex and NCNA moiety.

Aspect 22. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 20, wherein the NCNA moiety is between the targeting peptide complex and the PEG moiety.

Aspect 23. The targeted non-charged-nucleic acid (NCNA) construct of Aspects 16-22, further comprising a nuclear localization signal (NLS) moiety.

Aspect 24. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 23, wherein the NLS is attached to the NCNA on the opposite side of PEG, via a non-cleavable linker.

Aspect 25. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 16, wherein the targeting peptide complex and the NCNA moiety are linked via click chemistry.

Aspect 26. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 25, wherein either one, but not both, of the targeting peptide complex or the NCNA moiety is modified with an azide-containing moiety, and the other one of the targeting peptide complex or the NCNA moiety is modified with a reactive- alkyne moiety. Aspect 27. The targeted non-charged-nucleic acid (NCNA) construct of Aspect 25, wherein the targeting peptide complex is modified with azide-containing moiety, and wherein the NCNA moiety is modified with a reactive-alkyne moiety.

Aspect 28. The targeted non-charged-nucleic acid (NCNA) construct of Aspects 26-27, wherein the azide-containing moiety is AzidoAc, and the reactive-alkyne moiety is propargyl.

Aspect 29. A heterologous non-charged-nucleic acid (NCNA) conjugate capable of forming a tumor-penetrating-nanocomplex (TPN), comprising molecule selected from the following format formulas:

TPC-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-FA;

TPC-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); and y = 1 or 0 (e.g., present or absent); or

TPC- Lx - NCNA - Lx - EEM - Lx - FA;

TPC - Lx - EEM - Lx -NCNA - Lx - FA, wherein L is a linker; and x = 1 or 0; or

TPC-PEG-NCNA-EEM-FA;

TPC-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-PEG-FA;

TPC-PEG-EEM-PEG-NCNA-PEG-FA; or

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-NCNA-Lx-NLSz-Lx-PEGy-Lx-NL Sz-Lx-EEM-Lx-

NLSz-Lx-PEGy-Lx-NLSz-Lx-FA;

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-EEM-Lx-NLSz-Lx-PEGy-Lx-NLS z-Lx-NCNA-Lx-

NLSz-Lx-PEGy-Lx-NLSz-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); y = 1 or 0 (e.g., present or absent); and z = 1 or 0 (e.g., present or absent). Aspect 30. A Non-TPN forming heterologous non-charged-nucleic acid (NCNA) conjugate comprising molecule selected from the following format formulas:

TPC-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx;

TPC-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); and y = 1 or 0 (e.g., present or absent); or

TPC- Lx - NCNA - Lx - EEM - Lx

TPC - Lx - EEM - Lx -NCNA - Lx, wherein L is a linker; and x = 1 or 0; or

TPC-PEG-NCNA-EEM;

TPC-NCNA-PEG-EEM;

TPC-PEG-NCNA-PEG-EEM;

TPC-PEG-NCNA-PEG-EEM-PEG;

TPC-PEG-EEM-PEG-NCNA-PEG; or

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-NCNA-Lx-NLSz-Lx-PEGy-Lx-NL Sz-Lx-EEM-Lx-

NLSz-Lx-PEGy-Lx-NLSz-Lx;

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-EEM-Lx-NLSz-Lx-PEGy-Lx-NLS z-Lx-NCNA-Lx-

NLSz-Lx-PEGy-Lx-NLSz-Lx, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); y = 1 or 0 (e.g., present or absent); and z = 1 or 0 (e.g., present or absent).

Aspect 31. A method for treating a patient having a cancer, an immunodeficiency disorder, or an infection comprising

Brief Description of Drawings

[0008] Figures 1 A and IB show an exemplary TPN construct for delivery of a peptide nucleic acid (PNA). Fig. 1A corresponds to a molecule consisting of a targeting peptide, PNA, transportan, and fatty acid moieties. Fig. IB depicts a fatty-acid-driven self-assembly into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0009] Figures 2A and 2B show an exemplary TPN construct for delivery of a peptide nucleic acid (PNA) comprising a linker (starred). In Fig. 2A, a linker (cleavable or uncleavable) is shown between the targeting peptide-PNA moiety and the transportan-fatty acid moiety. Fig. 2B depicts a fatty acid-driven self-assembly into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0010] Figures 3 A and 3B show an exemplary TPN construct for delivery of a peptide nucleic acid (PNA) with a linker (starred). In Fig. 3A, a linker (cleavable or uncleavable) is shown between the targeting PNA moiety and the PNA-transportan-fatty acid moiety. Fig. 3B depicts a Fatty acid-driven assembly into a tumor-penetrating nanoparticle (TPN) in aqueous solution. [0011] Figures 4A and 4B illustrates an exemplary TPN construct for delivery of a peptide nucleic acid (PNA) comprising a plurality of linkers. In Fig. 4A, two linkers (cleavable or uncleavable) are present; the first linker between the targeting peptide moiety and the PNA moiety; and the second linker between the PNA moiety and the transportan-fatty acid moiety. Fig. 4B depicts a Fatty-acid-driven assembly into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0012] Figures 5 A and 5B show an exemplary TPN construct for delivery of a peptide nucleic acid (PNA) comprising a PEG, without a PEG, or a combination of PNAs both with a PEG and without a PEG. Fig. 5A and Fig. 5B depict Non-PEG-containing and PEG-containing molecules, respectively, comprising a targeting peptide, PNA, transportan, and fatty acid moieties. In this embodiment, the PEG moiety is located between the targeting peptide and PNA-transportan-fatty acid moieties. Fig. 5C depicts fatty acid-driven assembly into a tumorpenetrating nanoparticle (TPN) in aqueous solution.

[0013] Figures 6A and 6B show an exemplary TPN construct for delivery of a peptide nucleic acid (PNA) comprising a PEG, without a PEG, or a combination of PNAs with a PEG and without a PEG. Fig. 6A and Fig. 6B depict non-PEG-containing and PEG-containing molecules, respectively, comprising a targeting peptide, PNA, transportan, and fatty acid moieties. In this embodiment, the PEG moiety is located between the targeting peptide-PNA moiety and transportan-fatty acid moiety. Fig. 6C depicts fatty-acid-driven assembly into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0014] Figures 7A-7F show examples of non-fatty-acid-containing, peptide nucleic acid (PNA) conjugates of the present invention. In particular embodiments, the targeting peptide complex can comprise an iRGD/LSTAl -peptide.

[0015] Figure 8A-8F show other examples of non-fatty-acid-containing, peptide nucleic acid (PNA) conjugates of the present invention. In these Fig. 8A-8F embodiments, the locations of transportan and PNA are different from the examples illustrated in Figure 7. In particular embodiments, the targeting peptide complex can comprise an iRGD/LSTAl -peptide.

[0016] Figure 9 shows a process, set forth in Example 1 herein, for synthesizing an exemplary invention Non-TPN-Forming Conjugate resulting in the production of PNA-iRGD.

[0017] Figure 10 shows a process, set forth in Example 2 herein, for synthesizing an exemplary invention Non-TPN-Forming Conjugate resulting in the production of PNA-PEG- iRGD.

[0018] Figure 11 shows a process, set forth in Example 3 herein, for synthesizing an exemplary invention Non-TPN-Forming Conjugate resulting in the production of PEG-PNA- iRGD.

[0019] Figure 12 shows a process, set forth in Example 4 herein, for synthesizing an exemplary invention Non-TPN-Forming Conjugate resulting in the production of NLS-PNA- PEG-iRGD.

[0020] Figure 13 shows a process, set forth in Example 5 herein, for synthesizing an exemplary invention TPN-Forming Conjugate resulting in the production of FA-CPP-PNA- PEG-iRGD.

[0021] Figure 14 shows a process, set forth in Example 6 herein, for synthesizing an exemplary invention TPN-Forming Conjugate resulting in the production of FA-CPP-PEG- PNA-iRGD.

[0022] Figures 15A-15B show examples of heterologous non-charged-nucleic acid (NCNA) constructs comprising a single monoantennary targeting peptide complex (Fig. 15A-left panel); biantennary targeting peptide complex (Fig. 15 A-right panel); a triantennary targeting peptide complex (Fig. 15B-left panel); and a quadantennary targeting peptide complex (Fig. 15B-right panel).

[0023] Figures 16A and 16B show exemplary heterologous NCNA constructs and TPN constructs comprising a monovalent targeting moiety (also referred to herein as a monoantennary targeting peptide complex) for delivery of NCNAs. Fig. 16A shows both non- PEG-containing and PEG-containing molecules comprising a monovalently conjugated targeting peptide-NCNA, EEM (e.g., transportan, and the like), and fatty acid moieties. In this particular embodiment, the PEG moiety is located between the targeting peptide-NCNA moiety and the transportan-fatty acid moiety. Fig. 16B depicts fatty-acid-driven assembly of the NCNA constructs having a single, monoantennary targeting peptide complex, into an invention tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0024] Figures 17A and 17B show exemplary heterologous NCNA constructs and TPN constructs comprising a divalent targeting moiety (also referred to herein as a biantennary targeting peptide complex) for delivery of NCNAs. Fig. 17A shows both non-PEG-containing and PEG-containing molecules comprising a divalently conjugated targeting peptide-NCNA, EEM (e.g., transportan, and the like), and fatty acid moieties. In this particular embodiment, the PEG moiety is located between the targeting peptide-NCNA moiety and the transportan- fatty acid moiety. Fig. 17B depicts fatty-acid-driven assembly of the NCNA constructs having divalent, biantennary targeting peptide complex, into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0025] Figures 18A and 18B show exemplary heterologous NCNA constructs and TPN constructs comprising a trivalent targeting moiety (also referred to herein as a triantennary targeting peptide complex) for delivery of NCNAs. Fig. 18A shows both non-PEG-containing and PEG-containing molecules comprising a trivalently conjugated targeting peptide-NCNA, EEM (e.g., transportan, and the like), and fatty acid moieties. In this particular embodiment, the PEG moiety is located between the targeting peptide-NCNA moiety and the transportan- fatty acid moiety. Fig. 18B depicts fatty-acid-driven assembly of the NCNA constructs having trivalent, triantennary targeting peptide complex, into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

[0026] Figures 19A and 19B show exemplary heterologous NCNA constructs and TPN constructs comprising a quadrivalent targeting moiety (also referred to herein as a quadri antennary targeting peptide complex) for delivery of NCNAs. Fig. 19A shows both non- PEG-containing and PEG-containing molecules comprising a quadrivalently conjugated targeting peptide-NCNA, EEM (e.g., transportan, and the like), and fatty acid moieties. In this particular embodiment, the PEG moiety is located between the targeting peptide-NCNA moiety and the transportan-fatty acid moiety. Fig. 19B depicts fatty-acid-driven assembly of the NCNA constructs having quadrivalent, quadri antennary targeting peptide complex, into a tumor-penetrating nanoparticle (TPN) in aqueous solution.

Detailed Description

TPN-forming Targeted NCNA conjugate:

[0027] Provided herein is a heterologous non-charged-nuclei c acid (NCNA) conjugate capable of forming a tumor-penetrating-nanocomplex (TPN), comprising a targeting peptide complex (TPC); a non-charged-nucleic acid (NCNA); an endosome escape moiety (EEM); and a fatty acid moiety (FA), wherein the heterologous NCNA conjugate is capable of forming a tumor-penetrating- nanocomplex (TPN).

[0028] In particular embodiments, the various moieties desribed herein can be mixed and matached and interchangeably linked in different orders or formats. For example, in particular embodiments, the invention heterologous conjugates are depicted by the following format formulas:

TPC-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-FA;

TPC-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); and y = 1 or 0 (e.g., present or absent).

[0029] In other embodiments, the invention TPN forming conjugates can also be depicted as: TPC- Lx - NCNA - Lx - EEM - Lx - FA;

TPC - Lx - EEM - Lx -NCNA - Lx - FA, wherein L is a linker; and x = 1 or 0; and

TPC-PEG-NCNA-EEM-FA; TPC-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-PEG-FA;

TPC-PEG-EEM-PEG-NCNA-PEG-FA; and the like.

[0030] As set forth herein the NLS moiety can be added to any region of the invention heterologous NCNA conjugate as follows:

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-NCNA-Lx-NLSz-Lx-PEGy-Lx-NL Sz-Lx-EEM-Lx-NLSz- Lx-PEGy-Lx-NLSz-Lx-FA;

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-EEM-Lx-NLSz-Lx-PEGy-Lx-NLS z-Lx-NCNA-Lx-NLSz- Lx-PEGy-Lx-NLSz-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); y = 1 or 0 (e.g., present or absent); and z = 1 or 0 (e.g., present or absent).

Tumor-Penetrating Nanocomplex (TPN):

[0031] In particular embodiments, a plurality of invention NCNA conjugates combine to form a tumor-penetrating nanocomplex (TPN), via self-assembly of the respective fatty acid moieties therein as described in Figures 1-6, 13-14 and in Examples 5 and 6. Accordingly, also provided herein is are tumor-penetrating-nanocomplexs (TPNs), comprising the heterologous non-charged-nucleic acid conjugates described herein. The the heterologous non-charged- nucleic acid conjugates described herein and TPNs formed therefrom are useful in methods provided herein to treat a variety of disease including cancers, e.g, solid tumor cancers.

[0032] In particular embodiments, the invention TPN comprises a range of heterologous NCNA conjugates selected from: 2-10 ⁶ , 2-10 ⁵ , 2-10 ⁴ , 2-1000, 2-900, 2-800, 2-700, 2-600, 2- 500, 2-450, 2-400, 2-350, 2-300, 2-250, 2-200, 2-150, 2-125, 2-100, 2-90, 2-80, 2-70, 2-60, 2- 50 copies of the heterologous NCNA conjugate. In other embodiments, the invention TPN comprises an amount of heterologous NCNA conjugates selected from greater than: 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2-10 ⁴ , 2- 10 ⁵ , 2-10 ⁶ . In a particular embodiment, the invention TPN comprises 2-100 copies of the heterologous NCNA conjugate.

[0033] In particular embodiments, the invention TPN comprises 2 or more copies (e.g, a plurality) of the same heterologous NCNA conjugate. This particular TPN having a homogeneous population of heterologous NCNA conjugates is referred to herein as a homogenous TPN. It is also contemplated herein that the different heterologous NCNA conjugates set forth herein can be combined to form a TPN, referred to herein as heterogeneous TPN. Accordingly, in particular embodiments, it is contemplated herein that althought the targeting peptides within a single heterologous NCNA construct are the same/homogeneous, a plurality of different heterologous NCNA constructs can be used to form a particular hetergenous TPN, such that the TPN has a plural tiy of targets.

[0034] The invention NCNA constructs and TPNs formed thereby can incorporate in a variety of carriers, such as LNPs and silicon core fusogenic lipid nanoparticles. The ability to mix and match the different moieties and regions within the heterologous NCNA constructs, as set forth in Figures 1-19 herein, provides flexibility to address needs of a particular oligo and/or a particular target tissue.

Targeting-peptide Complex (TPC):

[0035] As used herein, the phrase "targeting peptide complex" refers to one peptide, or two or more (a plurality) targeting peptides tethered together in a single complex, that can be linked (either cleavably or non-cleavably) or conjugated to a non-charged-nucleic acid (NCNA), or an endosome escape moiety (EEM), or the like as described herein (see Figures 15-19).

[0036] As used herein, the phrase “monoantennary targeting peptide complex” refers to a targeting peptide complex having single targeting peptide (Figure 15(a)-left panel & Fig. 16).

[0037] As used herein, the phrase “ multi antennary targeting peptide complex” refers to a targeting peptide complex having more than one targeting peptide (Figure 15(a)-right panel; and (b) & Figs. 17-19).

[0038] In particular embodiments, the plurality of targeting peptides within a single targeting peptide complex are the same (e.g., homogeneous). In other embodiments, the plurality of targeting peptides within a single targeting peptide complex are not the same (e.g., heterogeneous). Accordingly, it is contemplated herein that multiple different targeting peptides can be used to

[0039] As used herein, the phrase “targeting peptide” or grammatical variations thereof, refers to any peptide region or moiety that functions to specifically localize the larger conjugate to which it is attached or associated, to a desired location, such as a target or antigen on a cell surface, and the like; and/or to facilitate cell penetration. In particular embodiments, the targeting peptide can target or direct a molecule or interest or the transport of the molecule (e.g., proteins, RNAs, DNAs, etc.) to a specific region, such as a region in a living body or a living tissue or in the cell. In another embodiment, the targeting peptides provide tumor and/or immune cell targeting and tissue/cell uptake.

[0040] In particular embodiments, the targeting peptides are no more than an amount selected from 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 amino acids in length. In another embodiment, the targeting peptides are no more than 40 amino acids in length. In another embodiment, the targeting peptides are no more than 30 amino acids in length. In another embodiment, the targeting peptides are no more than 20 amino acids in length.

[0041] In a particular embodiment, the targeting-peptide provides targeted-cell -peptide- penetration, referred to herein as targeted-CPP. In particular embodiments, the primary function of the targeting peptides used herein for targeted-CPP is to specifically localize the various moieties of the invention TPN-forming heterologous NCNA constructs/conjugates or non-TPN forming targeted NCNA constructs to specific cell-surfaces. Exemplary cell-surface targets that can be targeted for binding include, for example, any tumor or cancer cell. In particular embodiments, the tumor or cancer cells are from a solid tumor. In other embodiments, the tumor or cancer cells are from a liquid tumor.

[0042] Accordingly, exemplary targeting-peptides for use in targeting peptide complexes herein are those well-known in the art that bind, e.g., specifically bind, to antigens on solid tumors. In certain embodiments, targeting peptides for use herein can be found in, e.g., US 9,115,170; US 10,370,245; US 11,260,133, each of which are incorporated by reference in their entirety. Accordingly, exemplary targeting peptides contemplated for use herein have a length of less than about 100 residues and include the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:290 of US 9,115,170, one of the amino acid sequences CRGD(R/K/H)GV(D/E/H)C (SEQ ID NO:329 of US 9,115,170), or one of the amino acid sequences CRGDHGP(D/E/H)C (SEQ ID NO:330 of US 9,115,170) or a peptidomimetic thereof. Such an isolated targeting peptide can have, for example, a length of less than 50 residues or a length of less than 20 residues. In particular embodiments, the targeting peptide can include the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:290 of US 9,115,170, one of the amino acid sequences CRGD(R/K/H)GV(D/E/H)C (SEQ ID NO:329 of US 9,115,170), or one of the amino acid sequences CRGDHGP(D/E/H)C (SEQ ID NO:330 of US 9,115,170); or peptidomimetics thereof, and has a length of less than 10, 20, 50 or 100 residues.

[0043] In a particular embodiment, the targeting peptide is LSTA1 (CRGDKGPDC) corresponding to CAS Registry No: 2580154-02-3); or the iRGD peptide corresponding to CAS Registry No. 1392278-76-0. In other embodiments, targeting peptides for use herein comprise the amino acid sequence KRGARST or a peptidomimetic thereof, the amino acid sequence AKRGARSTA or a peptidomimetic thereof, or the amino acid sequence CKRGARSTC or a peptidomimetic thereof; each of which are disclosed in US 11,512,110, which is incorporated herein by reference in its entirety for all purposes. Also contemplated for use herein is a targeting peptide that binds to p32 at the cell surface referred to as LyP-1 (CGNKRTRGC; SEQ ID NO: 7 in US 11,512,110), or a peptidomimetic thereof.

[0044] NGR peptides are peptides that contain the NGR (Asn-Gly-Arg) motif and that home to angiogenesis and tumor vasculature. Examples of NGR peptides include CNGRCVSGCAGRC, NGRAHA, CVLNGRMEC, and CNGRC. GSL peptides are peptides that contain the GSL (Gly-Ser-Leu) motif and that home to tumor vasculature. Examples of a GSL peptide include CGSLVRC and CLSGSLSC.

[0045] Also contemplated herein are macrophage and/or dendritic cell-targeting peptides, that can be used, either with or without LSTA1 or iRGD co-administration. Exemplary targeting peptides that target Tumor-Associated Macrophages (TAMs) are set forth in US Patent 11,571,484; WO 2018/204392; and WO 2020/033663; each of which are incorporated herein by reference in their entirety for all purposes.

[0046] In one embodiment, peptides that target TAMs comprise SCPGAK or CSPGAKVRC and are no more than an amount selected from 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 amino acids in length. In another embodiment, peptides that target TAMs comprise one or more selected from RVLRSGS, GGRVLRS, RSGLRSS, GRLLRSG, GRMLRSG, GRVLRSS, or CRVLRSGSC; and are no more than an amount selected from 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 amino acids in length.

Non-Charged-Nucleic-Acid (NCNA): [0047] As set forth herein, the invention tumor-penetrating-nanocomplex (TPN) forming heterologous non-charged-nucleic acid (NCNA) conjugate comprises a non-charged nucleic acid (NCNA). As used herein, the phrase “non-charged nucleic acid (NCNA)” refers to a molecular structure that contains a nucleic acid chemically modified to be at least relatively non-charged or charge neutral compared to a naturally occurring form of the nucleic acid, which is polyanionic. For example, the nucleic acid can be conjugated with different types of molecules or can be chemically modified to be at least relatively non-charged or charge neutral. In particular embodiments, the NCNA contains nucleotide bases (e.g., DNA bases, and the like) attached to a non-charged backbone. The non-charged backbone can be in the form of peptide bonds, such as with peptide-nucleic acids (PNAs) or in the form of methylenemorpholine rings linked through phosphorodiamidate groups, such as with morpholinos, and the like.

[0048] In particular embodiments, the non-charged-nucleic acid (NCNA) is covalently attached to one or both of the targeting peptide complex and/or the endosome escape moiety by a linker. In certain embodiments, the linker is either cleavable or uncleavable.

[0049] As used herein, the term "linker" refers to a chemical moiety that contains two reactive group s/functional groups, one of which can react with one molecule and the other with another molecule. In some embodiments, the two reactive/functional groups of the linker can be linked via a linking moiety or spacer, structure of which is not critical as long as it does not interfere with the coupling of the linker to the two molecules.

1. Peptide Nucleic Acid (PNA)

[0050] Exemplary NCNAs for use herein include peptide nucleic acids (PNAs). PNAs are uncharged constructs with high binding affinity and other tunable properties. In PNAs, nucleotide bases (e.g., DNA with bases, RNA with bases, and the like) of a nucleic acid such as oligonucleotides can be attached to a non-charged backbone. It has been found that the neutral character of the PNA backbone can provide stronger binding between complementary PNA/DNA strands than between complementary DNA/DNA strands at low to medium ionic strength. This is attributed to the lack of charge repulsion between the PNA strand and the DNA strand. In addition, PNAs are not liable to enzymatic degradation and are less prone to non-specific binding than other oligo classes, and do not require protection from LNPs. Thus, in particular embodiments, PNAs avoid the need for lipid and/or PEG components. [0051] It is known that the Watson Crick base pairing rules are observed in hybrids of PNA and nucleic acids. Nielson & Egholm, Molec. Biol. 1 (1999) 1(2): 89-104. In certain embodiments, a PNA for use herein can comprise sequence information similar to DNA. PNAs are neither a peptide nor a nucleic acid or peptide-like structure. In further embodiments, exemplary PNAs include peptide nucleic acids with their polar or charged backbones (e.g., deoxyribose phosphodiester backbone) replaced by relatively less polar or non-charged backbones, such as a pseudo-peptide or amide backbone. The nucleic acids may be relatively homomorphous with the polar or charged backbone replaced.

[0052] In other embodiments, an exemplary PNA includes a structure comprising repeating N-(2-aminoethyl)-glycine units linked by amide bonds, a structure not containing any sugar moiety or phosphate group, and a structure comprising a purine (A or G) or pyrimidine (C or T) base attached to the non-charged backbone (e.g., attaching through methylene carbonyl linkages). An exemplary PNA includes homothymine PNAs, PNAs with purine bases, PNAs with pyrimidine bases, PNAs having backbones that are acyclic, achiral, nonpolar, non-charged or neural, PNAs capable of binding to complementary nucleic acids in antiparallel or parallel orientation, pseudo- complementary PNA containing diaminopurine-thiouracil, PNA oligomers, PNA conjugates (e.g., PNA-lipid conjugates), modified PNAs, and the like. See, e.g., Nielson & Egholm, Molec. Biol. 1 (1999) 1(2): 89-104; incorporated herein by reference in its entirety for all purposes.

[0053] In certain embodiments, exemplary PNAs include PNAs capable of forming three- dimensional structure such as three dimensional structure of four PNA complexes, hexamer duplexes (e.g., PNA-RNA duplex, PNA-PNA duplex, etc.), octamer duplexes (e.g., PNA-DNA duplex), triplexes (e.g., undecamer 2PNA/DNA triplexes, 2PNA/DNA triplexes, triplexes with bis-PNAs employed — in which the Watson-Crick PNA strand is connected by continuous synthesis via ethylene glycol type linkers to the Hoogsteen PNA strand). In another embodiment, an exemplary PNA includes molecules with backbones chemically modified, such as a glycine unit substituted by any of the amino acids; chemical modifications of aminoethyl glycine PNA backbone; and/or lysine derived backbones to which lysine-based monomers can be introduced. In yet a further embodiment, an exemplary PNA can include non-Watson-Crick nucleobases (e.g., pseudoisocytosine, diaminopurine, etc.).

[0054] In further embodiments, exemplary peptide-nucleic acids well-known in the art for use herein include the PNAs disclosed in US 7,820,624; US2009/0123467, and the like. 2. Phosphorodiamidate morpholino oligonucleotide (PMO) or Morpholinos

[0055] In other embodiments, the NCNA moiety includes various types of PMO (phosphorodiamidate morpholino oligonucleotide). As used herein, the phrase “phosphorodiamidate morpholino oligonucleotide (PMO)” or morpholino, or grammatical variations thereof, refers to a charge-neutral nucleic acid chemistry in which the five-membered ribose heterocycle is replaced by a six-membered morpholine; see, e.g., Langer, et al., Nat. Rev. Drug Discov. 19, 673-694 (2020). In particular embodiments, PMOs or “morpholinos” are nucleotide analogs that function to recognize and bind short sequences (e.g., about 25 nucleotides, and the like) at the transcription start site or at splice sites of pre-messenger RNAs (pre-mRNAs), and thereby to block the translation or proper splicing of the mRNA. In certain embodiments, PMO backbone linkages can contain chiral centers, meaning that PMOs can be racemic.

[0056] In particular embodiments, exemplary PMOs for use herein includes PMO-based steric block Antisense oligonucleotides (ASOs) and PMO conjugates, peptide-PMO (PPMO) conjugates (e.g., PPMO dystrophin, (RXR)4-PMO, etc ), B-MSP-PMO conjugates, Pip-PMO conjugates, CPP-PMOs, and the like.

3. Short interfering ribonucleic neutral (siRNN) chemistry molecules

[0057] A “ribonucleic acid” (RNA) is a polymer of nucleotides linked by a phosphodiester bond, where each nucleotide contains ribose or a modification thereof as the sugar component. Each nucleotide contains an adenine (A), a guanine (G), a cytosine (C), a uracil (U) or a modification thereof as the base. The genetic information in a mRNA molecule is encoded in the sequence of the nucleotide bases of the mRNA molecule, which are arranged into codons consisting of three nucleotide bases each. Each codon encodes for a specific amino acid of the polypeptide, except for the stop codons, which terminate translation (protein synthesis). Within a living cell, mRNA is transported to a ribosome, the site of protein synthesis, where it provides the genetic information for protein synthesis (translation).

[0058] The terms “RNAi agent,” “short interfering RNA”, “siRNA”, “short interfering nucleic acid”, “siNA” and the like as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference (RNAi) or gene silencing in a sequence-specific manner. The terms include short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), short interfering oligonucleotides, chemically-modified short interfering nucleic acid molecules, sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and/or tissues, RNAi agents wherein one or more mismatches exist between the sense and antisense strands, RNAi agents wherein the sense strand is very short relative to the antisense strand and/or has one or more single-stranded nicks, or any other molecule capable of mediating RNA interference. RNAi agents can comprise ribonucleotides, or be modified or substituted at one or more sugar, base and/or phosphate. As non-limiting examples, the RNAi agents can be modified at the 2' position with a 2'-modification selected from the group consisting of 2'- deoxy, 2 '-deoxy-2 '-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-0-M0E), 2'-O-aminopropyl (2'-0-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-O- DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-0-DMAE0E), and 2'-0 — N-methylacetamido (2'-0-NMA). In one embodiment, all pyrimidines (uridine and cytidine) are 2'-O-methyl- modified nucleosides. In various embodiments, one or more phosphate can be substituted with a modified intemucleoside linker, selected from phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, and an amide linker. In various embodiments, one or more nucleotides can be modified, or substituted with DNA, a peptide nucleic acid (PNA), locked nucleic acid (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), arabinose nucleic acid (ANA), 2'-fluoroarabinose nucleic acid (FANA), cyclohexene nucleic acid (CeNA), anhydrohexitol nucleic acid (HNA), unlocked nucleic acid (UNA). Various modifications and substitutions of RNAi agents are known in the art and can be used in the context of the instant disclosure. siRNAs are responsible for RNA interference, the process of sequence-specific post-transcriptional gene silencing in animals and plants. siRNAs are generated naturally by ribonuclease III cleavage from longer double-stranded RNA (dsRNA) which are homologous to, or specific to, the silenced gene target; artificial RNAi agents can be produced by any method known in the art.

[0059] In certain embodiments, exemplary NCNAs for use herein, include various shortinterfering ribonucleic neutral (siRNN) molecules. Reducing the overall charge on short interfering RNAs (siRNAs) while maintaining activity improves siRNA deliverability and stability. An exemplary siRNN includes phosphorothioates incorporated onto the ends of the siRNA strands (e.g., placing 6-8 phosphorothioates on the extended 3' single-stranded guide strand tail), yielding siRNA with reduced overall charge with neutral bioreversible phosphotriester oligonucleotides. See, e.g., Dowdy, Nat. Biotechnol. 35, 222-229 (2017). [0060] In other embodiments, exemplary siRNNs include siRNNs whose phosphate backbone contains neutral phosphotriester groups siRNNs conjugated to a targeting domain (see, e.g., Meade, et al. Nat. Biotechnol. 32, 1256-1261 (2014)). Also contemplated herein, is the use of a constrained DNA analogue (e.g., tricyclo-DNA (tcDNA)), as set forth in Roberts, T.C., Langer, et al., Nat Rev Drug Discov 19, 673-694 (2020).

Endosome Escape Moiety (EEM):

[0061] As set forth herein, the invention tumor-penetrating-nanocomplex (TPN) forming heterologous non-charged-nucleic acid (NCNA) conjugate comprises an endosome escape moiety (EEM). As used herein, the term “endosome escape moiety (EEM)” refers any moiety that functions to facilitate the release or escape of endosome contents from an edosome, or the escape of an invention TPN-forming conjugate molecule to which it is conjugated (or attached) from an endosome (or other internal cellular compartment). In other aspects, the endosome escape moiety (EEM) functions to avoid endosome sequestration for any molecule to which it is attached or conjugated. Accordingly, the EEM used herein can be any peptide moiety that is known in the art to penetrate and/or disrupt lipid bilayers. In certain embodiments, the EEMs for use herein are non-specific and promiscuous, such that the EEM will penetrate and/or disrupt any lipid bilayer. In particular embodiments, the lipid-bilayer penetrating activity of the EEM utilized in the invention TPN-forming-NCNA-conjugates is masked until the conjugate is within the endosome of a cell. In these embodiments, the cell-penetration of the invention TPN-forming conjuate is provided by the targeting peptide complex (e.g., the LSTA1 peptide). The masking of the EEM is achieved by incorporating the EEM moiety at particular regions within the TPN-forming NCNA conjugate (e.g, in between and targeting peptide complex and the NCNA or between the NCNA and the fatty acid or lipid moiety), such that when the conjugate enters the cell and is engulfed within an endosome (e.g., via endocytosis, and the like), the EEM’s lipid bilyer penetrating property is activated resulting in the NCNA payload of the TPN-forming conjugate being delivered into the cytosol to carry out its therapeutic function.

[0062] Examples of an endosome escape moiety include those disclosed in Dominska et al., Journal of Cell Science, 123(8): 1183-1189, 2010; Brock et al., Bioconjug Chem., 30(2): 293- 304, 2019 February 2019; U.S. Patent Publication 2019/0194655A1 to Curt W. Bradshaw et al. published on June 27 of 2019; U.S. Patent Publication 2017/0114341 Al to Curt W. Bradshaw et al. published on June 8 of 2015. The disclosures of these endosome escape moieties is incorporated by reference herein in their entirety.

[0063] Exemplary endosome escape moieties include polymers or molecules for facilitating or aiding endosome escape, such as peptides, lipids, nucleotides (e.g., endosomal escape domains, such as HA2, VSVG, GALA, Bl 8, etc.), cationic peptides such as cell-penetrating petides (CPPs) (e.g., transportan, TAT, and the like), amphiphilic peptides, cationic synthetic proteins cationic lipids, lipid nanoparticles (LNP), cationic polymers (polyethylenimine (PEI)), lipoplexes, lysosomotropic agents (e.g., L-leucyl-L-leucine O-methyl ester (LLOME), UNC7938, ). In figures 5-12 herein, the terms transportan and CPP are used interchangeably as more particular examples of the broader “endosome escape moiety” (EEM) genus.

[0064] In other embodiments, exemplary endosome escape moieties include chemotherapeutics (e.g., quinolones such as chloroquine), fusogenic lipids (e.g., dioleoylphosphatidyl-ethanolamine (DOPE)), poly(beta-amino ester)s, peptides or polypeptides such as cell-penetrating peptides, fusogenic peptides, polyarginines (e.g., octaarginine) and polylysines (e.g., octalysine), proton sponges, viral capsids, and peptide transduction domains as described herein. Exemplary endosome escape moieties include imidazole derivatives, histidine derivatives, poly(ethylenimine) and poly(L-histidine).

[0065] For example, fusogenic peptides can be derived from the M2 protein of influenza A viruses, peptide analogs of the influenza virus hemagglutinin, the HEF protein of the influenza C virus, the transmembrane glycoprotein of filoviruses, the transmembrane glycoprotein of the rabies virus, the transmembrane glycoprotein (G) of the vesicular stomatitis virus, the fusion protein of the Sendai virus, the transmembrane glycoprotein of the Semliki forest virus, the fusion protein of the human respiratory syncytial virus (RSV), the fusion protein of the measles virus, the fusion protein of the Newcastle disease virus, the fusion protein of the visna virus, the fusion protein of murine leukemia virus, the fusion protein of the HTL virus, and the fusion protein of the simian immunodeficiency virus (SIV). Other moieties that can be employed to facilitate endosome escape are described in Dominska et al., Journal of Cell Science, 123(8):1183-1189, 2010.

[0066] An exemplary endosome escape moiety can include a non-bioreversible linker attaching the endosome escape moiety to the conjugating moiety or a reaction product thereof (e.g., 1,2,3-triazole). The linker can be as described above for targeting moieties.

[0067] In particular embodiments, the endosome escape moiety facilitates the overcoming of endosomal entrapment of the respective molecule or composition comprising the EEM to allow for the release of cargos (e.g., macromolecular cargos) into the cytosol of cells. In more particular embodiment, the endosome escape moiety can be selected from one or more of: transportan, TAT peptide (RKKRRQRRR), poly-Y, and/or 6His-CM18-PTD4, a chimera of the 6His tag, PTD4 (YARAAAARQARA), and CM18 (KWKLFKKIGAVLKVLTTG).

[0068] In other embodiments, the endosome escape moiety can be selected from one or more of: CPPsite 2.0 (h ti s ://web ,i iitd . edu . in/raghav a/cp si te/) is an updated version of manually curated database (CPPsite) of cell-penetrating peptides (CPPs). The current version holds at least about 1850 peptide entries contemplated for use herein.

Fatty Acids (FA):

[0069] In further embodiments, the invention TPN-forming heterologous non-charged- nucleic acid (NCNA) conjugate further comprises a fatty acid moiety covalently attached to the endosome escape moiety. In particular embodiments, the fatty acid moiety is at the N- terminus of the conjugate. As used herein, the term "fatty acid" refers to a group of a long chain of hydrocarbon with a carboxylic acid at one end. Fatty acids and their associated derivatives are the primary components of lipids. In some embodiments, fatty acids can be hydrophobic moieties. A fatty acid may be represented by R-COOH, where R stands for the aliphatic moiety and COOH as the carboxylic group (making the molecule an acid). The general formula is CnFhn+ I COOH.

[0070] As used herein, the term “conjugated” refers to a bond association (e.g., covalent bond association) between two molecules to form a molecule. The term “conjugation” refers to the chemical reaction resulting in the bond association. In some embodiments, an exemplary conjugate includes the entity formed as a result of a covalent attachment of particular biomolecular moieties as described herein, such as a targeting peptide complex, a non-charged nucleie acid moiety (e.g., a PNA moiety, and the like), an edosome escape moiety (EEM; e.g., CPP, TAT, transportan, and the like), a PEG moiety and/or a fatty acid or phospholipid moiety, with or without the use of a linker as described herein. In some embodiments, a fatty acid conjugate or a lipid conjugate may inhibit aggregation of lipid particles (see., e.g., US8492359B2, incorporated herein by reference in its entirety).

[0071] In particular embodiments of the invention TPN-forming-NCNA-conjugates, fatty acid or lipid (phospholipid) moieties are conjugated with other moieties to increase the delivery of their conjugates to an intracellular environment (see, e.g., Figures 1-6). In various embodiments, lipid-conjugates can be comprised from at least one lipid moiety covalently bound to a monomer or polymeric moiety of either low or high molecular weight. When desired in particular embodiments, an optional bridging moiety can be used to link the fatty acid or phospholipid moiety to the remainder of the invention TPN-forming NCNA conjugates; e.g., to the EEM, such as transportan or a CPP, to the NCNA (e.g., PNA, and the like), or a PEG moiety. The conjugated moiety may be a low molecular weight carboxylic acid, dicarboxylic acid, fatty acid, dicarboxylic fatty acid, acetyl salicylic acid, cholic acid, cholesterylhemisuccinate, or mono- or di -saccharide, an amino acid or dipeptide, an oligopeptide, a glycoprotein mixture, a di- or trisaccharide monomer unit of a glycosaminoglycan such as a repeating unit of heparin, heparan sulfate, hyaluronic acid, chondrotin-sulfate, dermatan, keratan sulfate, or a higher molecular weight peptide or oligopeptide, a polysaccharide, polyglycan, protein, glycosaminoglycan, or a glycoprotein mixture. See, e.g., phospholipid-conjugates of high molecular weight, and associated analogues, in U.S. Pat. No. 5,064,817, as well as the publications cited herein. In other embodiments, exemplary fatty acids for use herein in conjugation to biomolecules are set forth in US US10786576B2, which is incorporated herein by reference for all purposes.

[0072] As used herein, the term “alkyl” refers to a fully saturated branched or unbranched (or straight chain or linear) hydrocarbon moiety, comprising 1 to 30 carbon atoms. In some embodiments, the alkyl comprises 5 to 20 carbon atoms, and in other embodiments, comprises 10 to 15 carbon atoms. C10-15alkyl refers to an alkyl chain comprising 10 to 15 carbons. The term “alkylene” refer to a divalent alkyl as defined supra.

[0073] The term “alkenyl” refers to a branched or unbranched hydrocarbon having at least one carbon-carbon double bond. The term “C2-30-alkynyl” refers to a hydrocarbon having two to seven carbon atoms and comprising at least one carbon-carbon triple bond.

[0074] The term “alkynyl” refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond. The term “C2-30-alkynyl” refers to a hydrocarbon having two to seven carbon atoms and comprising at least one carbon-carbon triple bond.

[0075] The term “aryl” refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6-10 carbon atoms in the ring portion. Representative examples of aryl are phenyl or naphthyl. [0076] The term “heteroaryl” includes monocyclic or bicyclic heteroaryl, containing from 5- 10 ring members selected from carbon atoms and 1 to 5 heteroatoms, and each heteroatoms is independently selected from O, N or S wherein S and N may be oxidized to various oxidation states. For bicyclic heteroaryl system, the system is fully aromatic (i.e. all rings are aromatic). [0077] The term “cycloalkyl” refers to saturated or unsaturated but non-aromatic monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms, preferably 3-8, or 3-7 carbon atoms. For bicyclic, and tricyclic cycloalkyl system, all rings are non-aromatic. For example, cycloalkyl encompasses cycloalkenyl and cycloalkynyl. The term “cycloalkenyl” refers to a bicyclic or tricyclic hydrocarbon group of 3-12 carbon atoms, having at least one carbon-carbon double bond. The term “cycloalkynyl” refers to a bicyclic or tricyclic hydrocarbon group of 3-12 carbon atoms, having at least one carbon-carbon triple bond.

[0078] The term “heterocyclyl” refers to a saturated or unsaturated non-aromatic (partially unsaturated but non-aromatic) monocyclic, bicyclic or tricyclic ring system which contains at least one heteroatom selected from O, S and N, where the N and S can also optionally be oxidized to various oxidation states. In one embodiment, heterocyclyl moiety represents a saturated monocyclic ring containing from 5-7 ring atoms and optionally containing a further heteroatom, selected from O, S or N. The heterocyclic ring may be substituted with alkyl, halo, oxo, alkoxy, haloalkyl, haloalkoxy. In other embodiment, heterocyclyl is di- or tricyclic. For polycyclic system, some ring may be aromatic and fused to saturated or partially saturated ring or rings. The overall fused system is not fully aromatic. For example, a heterocyclic ring system can be an aromatic heteroaryl ring fused with saturated or partially saturated cycloalkyl ring system.

[0079] In certain embodiments, when the conjugated carrier moiety is a polymer, the ratio of lipid moieties covalently bound may range from one to one thousand lipid residues per polymer molecule, depending upon the nature of the polymer and the reaction conditions employed. For example, the relative quantities of the starting materials, or the extent of the reaction time, may be modified in order to obtain Lipid-conjugate products with either high or low ratios of lipid residues per polymer, as desired.

[0080] In other embodiments, a lipid or phospholipid moiety can be selected from a: phosphatidic acid, an acyl glycerol, monoacylglycerol, diacylglycerol, triacylglycerol, sphingosine, sphingomyelin, chondroitin-4-sulphate, chondroitin-6-sulphate, ceramide, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, phosphatidylinositol, or phosphatidylglycerol; or an ether or alkyl phospholipid derivative thereof.

[0081] In further embodiments, the invention TPN-forming heterologous non-charged- nucleic acid (NCNA) conjugate further comprises a polyethylene-glycol (PEG) moiety covalently attached between the NCNA and targeting peptide complex and/or the NCNA and endosome escape moiety. Accordingly, the following format formulas are provided, although those of skill in the art will understand that the order of each moiety can readily be interchanged (e.g., mixed and matched):

TPC-PEG-NCNA-EEM-FA;

TPC-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-FA;

TPC-PEG-NCNA-PEG-EEM-PEG-FA;

TPC-PEG-EEM-PEG-NCNA-PEG-FA;

TPC-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-FA; TPC-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); and y present or absent.

[0082] As set forth herein the NLS moiety can be added to any region of the invention heterologous NCNA conjugate as follows:

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-NCNA-Lx-NLSz-Lx-PEGy-Lx-NL Sz-Lx-EEM-Lx-NLSz-

Lx-PEGy-Lx-NLSz-Lx-FA;

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-EEM-Lx-NLSz-Lx-PEGy-Lx-NLS z-Lx-NCNA-Lx-NLSz-

Lx-PEGy-Lx-NLSz-Lx-FA, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); y = 1 or 0 (e.g., present or absent); and z = 1 or 0 (e.g., present or absent).

[0083] Exemplary PEG moi eties for use herein include those set forth in US Patents: 9,493,499; 9,029,331, and the like. In particular embodiments, the poly(ethylene glycol) (PEG) moiety for use herein can have a molecular weight of up to about 100 kD. In other embodiments, exemplary PEG moi eties are approximately 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa or 80 kDa. The PEG moi eties are linear or branched PEG species, such as those described herein. In particular embodiments, the terminus of the PEG moiety, which is not attached to the linker, can be either OH or another moiety, e.g., O — (C1-C4) substituted or unsubstituted alkyl group. In one embodiment, OMe is presently preferred.

[0084] In further embodiments, exemplary PEG moieties for use herein include a methoxy PEG (mPEG) moiety, amine-terminated PEG (PEG-NH2) moiety, acetylated PEG (PEG-Ac) moiety, carboxylated PEG (PEG-COOH) moiety, thiol-terminated PEG (PEG-SH) moiety, N- hydroxy succinimide- activated PEG (PEG-NHS) moiety, NH2-PEG-NH2 moiety, and an NH2- PEG-COOH moiety. In particular aspects, the PEG moiety has a molecular weight including, but not limited to, a molecular weight of about 0.2 kDa to about 5 kDa. In some embodiments, the PEG moiety is a mPEG moiety with a molecular weight of about 2 kDa. In one embodiment, the PEG moiety is a mPEG moiety with a molecular weight of about 5 kDa.

Non-TPN-forming Targeted Heterologous NCNA constructs:

[0085] Also provided herein is a heterologous non-charged-nucleic acid (NCNA) construct, comprising a targeting peptide complex; a non-charged-nucleic acid (NCNA); and an endosome escape moiety (EEM).

[0086] Also provided herein is a targeted non-charged-nucleic acid (NCNA) construct, comprising: a targeting peptide complex; and a non-charged-nucleic acid moiety (NCNA).

[0087] Accordingly, the following Non-TPN forming conjugates or constructs are provided depicted by the following format formulas:

TPC-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx

TPC-Lx-PEGy-Lx-EEM-Lx-PEGy-Lx-NCNA-Lx-PEGy-Lx, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); and y = 1 or 0 (e.g., present or absent).

[0088] In other embodiments, the invention Non-TPN forming conjugates can also be depicted as:

TPC- Lx - NCNA - Lx - EEM - Lx;

TPC - Lx - EEM - Lx -NCNA - Lx wherein L is a linker; and x = 1 or 0; and

TPC-PEG-NCNA-EEM;

TPC-NCNA-PEG-EEM;

TPC-PEG-NCNA-PEG-EEM;

TPC-PEG-NCNA-PEG-EEM-PEG;

TPC-PEG-EEM-PEG-NCNA-PEG; and the like.

[0089] By virtue of the Non-TPN forming heterolgous NCNA conjugates lacking FA regions, these particular invention targeted NCNA constructs do not form a tumor penetrating nanocomplex described herein. In particular embodiments, as with other hNCNA conjugat constructs described herein, the targeting peptide complex and the NCNA are connected via a linker. The linker can be either cleavable or non-cleavable. In certain embodiments, the linker is non-cleavable.

[0090] In other embodiments of a targeted non-charged-nucleic acid (NCNA) construct, the targeting peptide complex and the NCNA moiety are connected as a single, contiguous polypeptide. In this embodiment, both the targeting peptide complex and the NCNA are synthesized contiguously as a single polypeptide in the same synthesis reaction, e.g. recombinantly produced using well-known genetic engineering methods.

[0091] In further embodiments, the invention targeted non-charged-nucleic acid (NCNA) construct further comprises a PEG moiety. In particular embodiments of the targeted NCNA construct, the PEG moiety is between the targeting peptide complex and NCNA moiety. In other embodiments, the NCNA moiety is between the targeting peptide complex and the PEG moiety.

[0092] In yet further embodiments, the invention targeted non-charged-nucleic acid (NCNA) construct further comprises a nuclear localization signal (NLS) moiety. In particular embodiments, the NLS is attached to the NCNA on the distal or opposite side of PEG, via a non-cleavable linker.

[0093] As with the TPN-forming conjugates described herein, an NLS moiety can be added to any region of the invention Non-TPN forming heterologous NCNA conjugate as follows:

TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-NCNA-Lx-NLSz-Lx-PEGy-Lx-NL Sz-Lx-EEM-Lx-NLSz- Lx-PEGy-Lx-NLSz-Lx; TPC-Lx-NLSz-Lx-PEGy-Lx-NLSz-Lx-EEM-Lx-NLSz-Lx-PEGy-Lx-NLSz-L x-NCNA-Lx-NLSz- Lx-PEGy-Lx-NLSz-Lx, wherein L is a linker; and x = 1 or 0 (e.g., present or absent); y = 1 or 0 (e.g., present or absent); and z = 1 or 0 (e.g., present or absent).

[0094] As used herein, the phrase “nuclear localization signal (NLS)” refers to short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus. This NLS-dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex, is facilitated by members of the importin superfamily. The NLS can be attached to any moiety within an invention TPN- forming or Non-TPN-forming heterologous NCNA conjugate described herein, including via a cleavalbe or non-cleavable linker.

[0095] Exemplary NLSs are set forth, for example, in Lu et al., Cell Communication and Signaling, Vo. 19, Article number: 60 (2021), which is incorporated herein by reference in its entirety for all purposes; and include in Table 1, among others: simian virus 40 (SV40) NLS, Pro-Lys-Lys-Lys-Arg-Lys-Val (PKKKRKV);

VACM-1/CUL5 NLS: PKLKRQ;

CXCR4 NLS RPRK;

VP1 NLS: RRARRPRG;

53BP1 NLS: GKRKLITSEEERSPAKRGRKS;

ING4 NLS: KGKKGRTQKEKKAARARSKGKN;

IERS NLS: RKRCAAGVGGGPAGCPAPGSTPLKKPRR;

ERRS NLS: RKPVTAQERQREREEKRRRRQERAKEREKRRQERER;

Hrpl NLS: RSGGNHRRNGRGGRGGYNRRNNGYHPY;

UL79 NLS: TLLLRETMNNLGVSDHAVLSRKTPQPY;

EWS NLS: PGKMDKGEHRQERRDRPY; PTHrP NLS: GKKKKGKPGKRREQRKKKRRT;

Pho4 NLS: SANKVTKNKSNSSPYLNKRKGKPGPDS; rpL23a NLS: VHSHKKKKIPTSPTFTTPKTLTLRRQPKYPRKSAPRRNKLDHY;

MSX1 NLS: RI<HI<TNRI<PR & NRRAKAKR;

NLS-RARa: RNI<I<I<I< & RKVIK.

Click Chemistry Linker:

[0096] In particular embodiments, the targeting peptide complex and the NCNA moiety are non-cleavably linked using the well-known click chemistry (e.g., a bioorthogonal reaction). Bioorthogonal reactions are reactions of materials with each other, wherein each material has limited or substantially no reactivity with functional groups found in vivo. The efficient reaction between an azide and a terminal alkyne, is a well-known implementation of “click” chemistry, bioorthogonal reaction. In particular embodiments, the Cu(I) catalyzed 1,3-dipolar cyclization of azides with terminal alkynes to give stable triazoles (see e.g., US 8,580,970; and Binder et al., Macromol. Rapid Commun. 2008, 29:952-981; each of which are incorporated be reference in their entirety for all purposes).

[0097] In particular embodiments, a process is utilized for catalyzing a click chemistry ligation reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety, as set forth in US 8,580,970, and the like. More particularly, the click chemistry ligation reaction is catalyzed by an addition of Cu(II) in the presence of a reducing agent for reducing said Cu(II) to Cu(I), in situ, in catalytic amount. Preferred reducing agents include ascorbate, metallic copper, quinone, hydroquinone, vitamin KI, glutathione, cysteine, Fe2+, Co2+, and an applied electric potential. Further preferred reducing agents include metals selected from the group consisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.

[0098] In alternative embodiments, a click chemistry ligation reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety is catalyzed by performing the click chemistry ligation reaction in a solution in contact with metallic copper. The metallic copper contributes directly or indirectly to the catalysis of the click chemistry ligation reaction. In a preferred mode, the solution is an aqueous solution. The first and second reactants may be present during the click chemistry ligation reaction in equimolar amounts. Also, the click chemistry ligation reaction may be performed in a solution in contact, at least in part, with a copper vessel.

[0099] In yet another embodiment, a click chemistry ligation reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety is catalyzed by an addition of a catalytic amount of a metal salt having a metal ion selected from the group consisting of Au, Ag, Hg, Cd, Zr, Ru, Fe, Co, Pt, Pd, Ni, Rh, and W. In a preferred mode of this aspect of the invention, the click chemistry ligation reaction is performed in the presence of a reducing agent for reducing said metal ion to a catalytically active form. Preferred reducing agents include ascorbate, quinone, hydroquinone, vitamin KI, glutathione, cysteine, Fe2+, Co2+, an applied electric potential, and a metal selected from the group consisting of Al, Be, Co, Cr, Fe, Mg, Mn, Ni, and Zn.

[00100] Numerous other mechanisms of click chemistry are also contemplated for use herein, including for example, those described in Chem. Rev. 2021, 6697-6698, which are each incorporated herein by reference in its entirety for all purposes. These click chemistries include the following types of biomolecular applications of click chemistry: Brown et al. teaches the use of click reactions with nucleic acids (Chem. Rev. 2021, 121, 12, 7122-7154); Paegel discloses the use of a variety of reactions in the presence of DNA, the key chemical requirement to harness the power of DNA-encoded libraries (Chem. Rev. 2021, 121, 12, 7155-7177); and Suazo et al., Chem. Rev. 2021, 121, 12, 7178-7248, teaches the use of click chemistry for the modification of lipids and carbohydrates

[00101] In other embodiments, contemplated for use herein are the reversible, cleavable click chemistry mechanisms set forth in Shieh et al., Chem. Rev. 2021, 121, 12, 7059-712 (incorporated herein be reference in its entirety for all purposes), which mechanism provides selective and efficient covalent bond breaking reactions (also referred to as “clip reactions”).

[00102] In particular embodiments of the targeted non-charged-nuclei c acid (NCNA) construct, either one, but not both, of the targeting peptide complex or the NCNA moiety is modified with an “azide-containing moiety”, and the other one of the targeting peptide complex or the NCNA moiety is modified with a “reactive-alkyne moiety”. In other embodiments, either one, but not both, of the targeting peptide complex or the NCNA moiety is modified with a AzidoAc, and the other one of the targeting peptide complex or the NCNA moiety is modified with a propargyl. In certain embodiments, the targeting peptide complex is modified with anAzido-containing compound and the NCNA moiety is modified with a reactive-alkyne. In yet another embodiment, the targeting peptide complex is modified with an AzidoAc, and wherein the NCNA moiety is modified with a propargyl.

[00103] As used herein, the phrase “azide-containing moiety” refers to moiety containing a linear, polyatomic anion with the structure -N=N+=N- It is the conjugate base of hydrazoic acid HN3. Organic azides are organic compounds with the formula RN3, containing the azide functional group. The azide anion behaves as a nucleophile; it undergoes nucleophilic substitution for both aliphatic and aromatic systems.

[00104] As used herein, the phrase “reactive-alkyne moiety” refers to a terminal alkyne that is an acyclic (either branched or unbranched) aliphatic hydrocarbon having one carbon-to- carbon triple bond; e.g., having the general molecular formula CnH2n-2. Exemplary reactive alkynes indlude for use herein, include among others, propargyl. In a particular embodiment, the reactive terminal alkyne used herein is porpargyl.

[00105] As set forth herein, in particular embodiments, a process is utilized for catalyzing a click chemistry ligation reaction between a first reactant having a terminal alkyne moiety and second reactant having an azide moiety for forming a product having a triazole moiety, as set forth in US 8,580,970, and the like.

[00106] As set forth herein, in particular embodiments, the linker separates the NCNA construct moieties, e.g., the fatty acid moiety from a transportan moiety. Its chemical structure is not critical, since it serves primarily as a spacer.

[00107] The linker can be made up of amino acids linked together by peptide bonds. In some embodiments of the present invention, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. In various embodiments, the 1 to 20 amino acids are selected from the amino acids glycine, serine, alanine, methionine, asparagine, glutamine, cysteine and lysine. In some embodiments, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. In some embodiments, linkers are polyglycines, polyalanines, combinations of glycine and alanine (such as poly(Gly-Ala)), or combinations of glycine and serine (such as poly(Gly-Ser)). In some embodiments, a linker is made up of a majority of amino acids selected from histidine, alanine, methionine, glutamine, asparagine and glycine. In some embodiments, linkers contain poly-histidine moiety.

[00108] In some embodiments, the linker comprises 1 to 20 amino acids which are selected from unnatural amino acids. While a linker of 1-10 amino acid residues is preferred for conjugation with the fatty acid moiety, the present invention contemplates linkers of any length or composition. An example of non-natural amino acid linker is 8-Amino-3,6-dioxaoctanoic acid having the following formula: or its repeating units.

[00109] The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention. Non-peptide linkers are also contemplated by the present invention.

[00110] In other embodiments, the linker comprises one or more alkyl groups, alkenyl groups, cycloalkyl groups, aryl groups, heteroaryl groups, heterocyclic groups, polyethylene glycol and/or one or more natural or unnatural amino acids, or combination thereof, wherein each of the alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, polyethylene glycol and/or the natural or unnatural amino acids are optionally combined and linked together, or linked to the biomolecule and/or to the fatty acid moiety, via a chemical group selected from — C(O)O — , — OC(O)— , — NHC(O)— , — C(O)NH— , — O— , — NH— , — S— , — C(O)— , — OC(O)NH— , — NHC(O)— O— , =NH— O— , =NH— NH— or =NH— N(alkyl)-.

[00111] Linkers containing alkyl spacer are for example — NH — (CH2)z — C(O) — or — S — (CH ₂ )/— C(O)— or — O— (CH ₂ )z— C(O)— , — NH— (CH ₂ ) _Z — NH— , — O— C(O)— (CH ₂ ) _Z — C(O)— O— , — C(O)— (CH ₂ ) _Z — O— , — NHC(O)— (CH ₂ ) _Z — C(O)— NH— and the like wherein z is 2-20 can be used. These alkyl linkers can further be substituted by any non- sterically hindering group, including, but not limited to, a lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH ₂ , or phenyl.

[00112] The linker can also be of polymeric nature. The linker may include polymer chains or units that are biostable or biodegradable. Polymers with repeat linkage may have varying degrees of stability under physiological conditions depending on bond lability. Polymers may contain bonds such as polycarbonates ( — O — C(O) — O — ), polyesters ( — C(O) — O — ), polyurethanes ( — NH — C(O) — O — ), polyamide ( — C(O) — NH — ). These bonds are provided by way of examples, and are not intended to limit the type of bonds employable in the polymer chains or linkers of the invention. Suitable polymers include, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-hydroxypropyl)-methacrylicamide, dextran, dextran derivatives, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether, and the like and mixtures thereof. A polymer linker is for example polyethylene glycol (PEG). The PEG linker can be linear or branched. A molecular weight of the PEG linker in the present invention is not restricted to any particular size, but certain embodiments have a molecular weight between 100 to 5000 Dalton for example 500 to 1500 Dalton.

[00113] The linker contains appropriate functional-reactive groups at both terminals that form a bridge between, for example, the amino group of the peptide or polypeptide/protein and a functional/reactive group on the fatty acid moiety, and the like.

[00114] Reactive groups of particular interest for conjugating a biomolecule or modified biomolecule to a linker and/or a linker to the fatty acid moiety and/or to conjugate various linking moieties of different nature together are N-hydroxysuccinimide, alkyne (more particularly cyclooctyne). While several examples of linkers and functionalities/reactive group are described herein, the invention contemplates linkers or any length and composition.

Methods of Treatment

[00115] Provided herein are methods for treating a patient having a cancer, an immunodeficiency disorder, or an infection comprising:

[00116] (a) administering to the patient a dose of the TPN-forming heterologous NCNA conjugate; an invention TPN; or the non-TPN forming NCNA constructs to a patient in need thereof.

[00117] An invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, can be administered to human patients to treat a variety of conditions. Optionally, such therapies can be administered parenterally, although oral routes may be possible if the therapeutic is formulated specifically to make oral administration possible without destruction of the therapeutic in the acid environment of the stomach. In some embodiments, such therapeutics can be administered by injection, optionally, for example, by intramuscular, subcutaneous, intravenous, intraarterial, intradermal, or intratumoral injection. Injections can be administered by infusion or in a bolus. In some embodiments, administration of the therapeutic can occur through a mucosal membrane. Such routes of administration include, e.g., nasal, rectal, or vaginal administration or administration under the eyelids or the tongue (without swallowing) or via inhalation. [00118] A dose of an invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, can be at least 0.1 mg/kg and not more than 5, 10, or 15 mg/kg. In some embodiments, the dosage can be less than or equal to 10, 8, 5, 3, 2, or 1 mg/kg and/or at least 0.1, 0.3, 1, 2, or 3 mg/kg. In some embodiments, the dosage can be about 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, or 9 mg/kg. Further a dosage may be defined as a specific amount, independent of the weight of the patient. Such doses can range from about 5 mg to about 800 mg. In particular embodiments, such a dose can be no more than 800, 700, 600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 100, 75, or 50 mg and/or at least about 60, 80, 100, 150, 200, 250, or 300 mg. Further, a dose can be about 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, or 450 mg. Alternatively, a dosage can be defined relative to the surface area of the skin of a patient. For example, in some embodiments a dosage can be at least 3.5 mg/mm ² and not more than 180 mg/mm ² . In some embodiments, a dose can be no more than 400, 350, 300, 350, 200, 180, 150, 110, 75, 50, 40, 30, 25, 12, 10, 7.5, or 5 mg/mm ² and/or at least 0.2, 0.5, 1, 3, 5, 10, 20, 30, 50, 75, or 100 mg/mm ² .

[00119] Doses of an invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, can be at least 0.033 mg/kg and not more than 3.35, 6.7, or 10 mg/kg. In some embodiments, the dosage can be less than or equal to 10, 6.7, 4.8, 3.35, 2, or 0.67 mg/kg and/or at least 0.033, 0.1, 0.33, 0.67, or 1 mg/kg. In some embodiments, the dosage can be about 0.033 mg/kg, 0.1 mg/kg, 0.67 mg/kg, 1.0 mg/kg, 1.67 mg/kg, 3.0 mg/kg, 3.33 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, or 10 mg/kg. Further a dosage may be defined as a specific amount, independent of the weight of the patient. Such doses can range from about 60 mg to about 700 mg. In particular embodiments, such a dose can be no more than 700, 600, 500, 450, 400, 350, 300, 250, 215, 170, 130, 100, or 70 mg and/or at least about 0.033, 0.1, 0.7, 1.5, 2, 2.7, 3.5, 5, 7, 10, 15, 17, 20, 25, 35, 45, 55, 65, 100, or 150 mg. Alternatively, a dosage can be defined relative to the surface area of the skin of a patient. For example, a dosage can be at least 0.1, 0.5, or 1.1 mg/mm ² and not more than 350, 300, 250, 200, or 130 mg/mm ² . In some embodiments, a dose can be no more than 300, 200, 130, 120, 100, 75, 50, 35, 30, 20, 15, 10, 7.5, 5, or 3 mg/mm ² and/or at least 0.18, 0.3, 1, 2, 3, 6, 10, 17, 25, 33, 66, or 80 mg/mm ² .

[00120] The frequency of dosing in amounts discussed above can be adjusted. In some embodiments an invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, can be administered once every three weeks. In other embodiments, such therapeutics can be administered twice per week, once per week, once every 10 days, once every two weeks, or once every three, four, five, six, seven, eight, nine, or 10 weeks. In further embodiments, such therapeutics can be administered once every two, three, four, five, six, seven, eight, nine, 10, 11, or 12 months.

[00121] An an invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, can be used to treat human patients having a variety of conditions. Since such therapeutics are contemplated herein to enhance some aspects of an immune response, the conditions for which they are a useful generally include conditions where an enhanced immune response is beneficial. The conditions treatable with the above-mentioned therapeutics include infections, immunodeficiency disorders, and various cancers including, without limitation, melanoma, lung cancer, including squamous non-small cell lung cancer and small cell lung cancer, nasopharyngeal cancer, squamous cell carcinoma of the head and neck, gastric or gastroesophageal carcinoma, clear cell or non-clear cell renal cell carcinoma, urothelial cancer, soft tissue or bone sarcoma, mesothelioma, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, bladder cancer, Merkel cell carcinoma, neuroendocrine carcinoma, cervical cancer, hepatocellular carcinoma, ovarian cancer, microsatellite instability high (MSI- H) or DNA mismatch repair deficient (dMMR) adult and pediatric solid tumors, clear cell renal sarcoma, colorectal cancer, esophageal cancer including esophageal squamous cell carcinoma, endometrial cancer, tumor mutational burden-high cancer, and cutaneous squamous cell carcinoma.

[00122] In particular embodiments, the invention heterologous NCNA conjugate or construct, of inventionn TPNs formed therefrom, are particularly useful for treaing solid tumor cancers. Exemplary solid tumors that can be treated herein include pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, including metastatic breast cancer, prostate cancer, including androgen-dependent and androgen-independent prostate cancer, renal cancer, including, e.g., metastatic renal cell carcinoma; hepatocellular cancer, lung cancer, including, e.g., non-Small cell lung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, e.g., progressive epithelial or primary peritoneal cancer, cervical cancer; gastric cancer, esophageal cancer, head and neck cancer, including, e.g., squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, e.g., glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer, and soft tissue sarcoma, brain and other central nervous system tumors (e.g. tumors of the meninges, brain, spinal cord, cranial nerves and other parts of central nervous system, e.g. glioblastomas or medulla blastomas); head and/or neck cancer; breast tumors; circulatory system tumors (e.g. heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue); excretory system tumors (e.g. kidney, renal pelvis, ureter, bladder, other and unspecified urinary organs); gastrointestinal tract tumors (e.g. oesophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus and anal canal), tumors involving the liver and intrahepatic bile ducts, gall bladder, other and unspecified parts of biliary tract, pancreas, other and digestive organs); head and neck; oral cavity (lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx); reproductive system tumors (e.g. vulva, vagina, Cervix uteri, Corpus uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); respiratory tract tumors ( e.g. nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung, e.g. small cell lung cancer or non-small cell lung cancer); skeletal system tumors (e.g. bone and articular cartilage of limbs, bone articular cartilage and other sites); skin tumors (e.g. malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eye and adnexa, thyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites.

Examples

Example 1 - Non-TPN-Forming Conjugates (a)(Fig. 9)

[00123] As depicted in Figure 9, using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized containing a succinimidyl valeric acid (SV A) functional group. This PNA-SVA is then purified using standard chromatographic techniques and isolated as a lyophilized solid at -20 °C. PNA-SVA is then coupled to BCN- amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl] -1.8-diamino-3,6- dioxaoctane) to yield PNA-Propargyl. Then, via standard copper-free click chemistry, PNA- Propargyl is reacted with azidoacetyl -iRGD to yield PNA-iRGD.

Example 2 - Non-TPN-Forming Conjugates (b)(Fig. 10)

[00124] As depicted in Figure 10, Uuing standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized; the PNA sequence contains a cysteine analogue, which contains a free thiol (-SH) group. This PNA-Cys-SH is then purified using standard chromatographic techniques and isolated as a lyophilized solid at -20 °C. PNA-Cys- SH is then reacted with a heterobifunctional polyethylene glycol) (average molecular weight of -5,000 Da), OPSS-PEG5k-SVA, to form a disulfide bond and yield PNA-S-S-PEG-SVA. PNA-S-S-PEG-SVA is then coupled to BCN-amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn- 9-ylmethyloxycarbonyl]-1.8-diamino-3,6-dioxaoctane) to yield PNA-Propargyl. Then, via standard copper-free click chemistry, PNA-Propargyl is reacted with azidoacetyl -iRGD to yield PNA-iRGD.

Example 3 - Non-TPN-Forming Conjugates (c)(Fig. 11)

[00125] As depicted in Figure 11, using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized; the PNA sequence contains a cysteine analogue, which contains a free thiol (-SH) group, and also contains an internalizing RGD motif. This Cys-PNA-iRGD is then purified using standard chromatographic techniques and isolated as a lyophilized solid at -20 °C. Cys-PNA-iRGD is then reacted with a monofunctional (thiol-containing) polyethylene glycol) mPEG5k-SH, to form a disulfide bond and yield PEG- PNA-iRGD. Example 4 - Non-TPN-Forming Conjugates (d)(Fig. 12)

[00126] As depicted in Figure 12, using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized; the PNA sequence contains a nuclear localization sequence (NLS) as well as a cysteine analogue, which contains a free thiol (-SH) group. This NLS-PNA-Cys-SH is then purified using standard chromatographic techniques and isolated as a lyophilized solid at -20 °C. This NLS-PNA-Cys-SH is then reacted with a heterobifunctional poly(ethylene glycol) (average molecular weight of -5,000 Da), OPSS- PEG5k-SVA, to form a disulfide bond and yield NLS-PNA-S-S-PEG-SVA. NLS-PNA-S-S- PEG-SVA is then coupled to BCN-amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9- ylmethyloxy carbonyl]- 1.8-diamino-3,6-di oxaoctane) to yield NLS-PNA-S-S-PEG-Propargyl. Then, via standard copper-free click chemistry, NLS-PNA-S-S-PEG-Propargyl is reacted with azidoacetyl-iRGD to yield NLS-PNA-PEG-iRGD.

Example 5 - TPN-Forming Conjugates (a)(Fig. 13)

[00127] As depicted in Figure 13-(Component 2a-l), using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized. The synthesized molecule also contains a terminal fatty acid (FA), such as palmitoyl or myristoyl, as well as a cell-penetrating peptide (CPP) motif (such as transportan, and the like), and a succinimidyl valeric acid (SVA) functional group. This molecule is then purified using standard chromatographic techniques and isolated as a lyophilized solid (FA-CPP-PNA-SVA) at -20 °C. This FA-CPP-PNA-SVA is then coupled to BCN-amine (N-[(lR,8S,9s)- Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1.8-diamino-3, 6-dioxaoctane) to yield FA- CPP-PNA-Propargyl. Then, via standard copper-free click chemistry, FA-CPP-PNA- Propargyl is reacted with azidoacetyl-iRGD to yield FA-CPP-PNA-iRGD.

[00128] As depicted in Figure 13-(Component 2a-2), using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized; the PNA sequence contains a cysteine analogue, which contains a free thiol (-SH) group. The synthesized molecule also contains a terminal fatty acid (FA), such as palmitoyl or myristoyl, as well as a cell-penetrating peptide (CPP) motif, such as transportan. This molecule is then purified using standard chromatographic techniques and isolated as a lyophilized solid (FA-CPP-PNA-Cys- SH) at -20 °C. FA-CPP-PNA-Cys-SH is then reacted with a heterobifunctional poly(ethylene glycol) (average molecular weight of -5,000 Da), OPSS-PEG5k-SVA, to form a disulfide bond and yield FA-CPP-PNA-S-S-PEG-SVA (FA-CPP-PNA-PEG-SVA). FA-CPP-PNA-PEG- SVA is then coupled to BCN-amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9- ylmethyloxy carbonyl]- 1.8-diamino-3,6-di oxaoctane) to yield FA-CPP-PNA-PEG-Propargyl. Then, via standard copper-free click chemistry, FA-CPP-PNA-PEG-Propargyl is reacted with azidoacetyl -iRGD to yield FA-CPP-PNA-PEG-iRGD.

[00129] Aqueous solutions of FA-CPP-PNA-iRGD and FA-CPP-PNA-PEG-iRGD are then combined to yield tumor-penetrating nanocomplexes (TPNs) via self-assembly of the fatty acid moieties.

Example 6 - TPN-Forming Conjugates (b)(Fig. 14)

[00130] As depicted in Figure 14-(Component 2b-l), Using standard solid-phase peptide chemistry, a peptide nucleic acid (PNA) of desired sequence is synthesized. The synthesized molecule also contains a terminal fatty acid (FA), such as palmitoyl or myristoyl, as well as a cell-penetrating peptide (CPP) motif (such as transportan), and a succinimidyl valeric acid (SV A) functional group. This molecule is then purified using standard chromatographic techniques and isolated as a lyophilized solid (FA-CPP-PNA-SVA) at -20 °C. This FA-CPP- PNA-SVA is then coupled to BCN-amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9- ylmethyloxy carbonyl]- 1 ,8-diamino-3,6-di oxaoctane) to yield FA-CPP-PNA-Propargyl. Then, via standard copper-free click chemistry, FA-CPP-PNA-Propargyl is reacted with azidoacetyl- iRGD to yield FA-CPP-PNA-iRGD.

[00131] As depicted in Figure 14-(Component 2b-2), using standard solid-phase peptide chemistry, a molecule is synthesized which contains a terminal fatty acid (FA), such as palmitoyl or myristoyl, as well as a cell-penetrating peptide (CPP) motif, such as transportan, and a cysteine residue which contains a free thiol (-SH) group. .This molecule is then purified using standard chromatographic techniques and isolated as a lyophilized solid (FA-CPP-Cys- SH) at -20 °C. FA-CPP-Cys-SH is then reacted with a heterobifunctional poly(ethylene glycol) (average molecular weight of -5,000 Da), OPSS-PEG5k-SVA, to form a disulfide bond and yield FA-CPP-S-S-PEG-SVA (FA-CPP-PEG-SVA). FA-CPP-PEG-SVA is then coupled to BCN-amine (N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl] -1.8-diamino- 3,6-dioxaoctane) to yield FA-CPP-PEG-Propargyl. Then, via standard copper-free click chemistry, FA-CPP-PNA-PEG-Propargyl is reacted with azidoacetyl-PNA-iRGD to yield FA- CPP-PEG-PNA-iRGD.

[00132] Aqueous solutions of FA-CPP-PNA-iRGD and FA-CPP-PEG-PNA-iRGD are then combined to yield tumor-penetrating nanocomplexes (TPNs) via self-assembly of the fatty acid moieties.