CD22 receptor-mediated delivery of polynucleotides

Abstract

A composition comprising a complex containing a polynucleotide of interest and a carrier is provided. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand and can target CD22 on cells. The carrier may contain a polymer, a lipid, or a protein. The polynucleotide of interest may contain, for example, plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), or small interfering RNA (siRNA) that alters level or activity of a target molecule in B cells. Methods of delivering a polynucleotide of interest to a CD22 expressing cell, and methods of treating a B cell-associated disease (such as acute lymphoblastic leukemia) in a subject using the composition are also provided.

Description

Attorney Docket No: LLU 24-009 (105781.0238.0)CD22 RECEPTOR-MEDIATED DELIVERY OF POLYNUCLEOTIDESInventors: Venkata Sesha Sai Abhinav Ayyadevara, Christian HurtzCROSS-REFERENCE TO RELATED APPLICATION

[0001] This claims priority to, and benefit of, U.S. Provisional Patent Application Serial No. 63 / 715,667 filed November 04, 2024. The entire contents of the above application are hereby incorporated by reference.TECHNICAL FIELD

[0002] The disclosure relates to cell-targeted delivery of therapeutic molecules.BACKGROUND

[0003] B cell associated diseases, such as acute lymphoblastic leukemia (ALL or B-ALL) in patients, are currently treated by antibody-drug conjugate therapy, CAR-T therapy, or chemotherapeutic or pharmacologic drugs. However, none of these therapeutic interventions offers a cure and shows toxic side effects. These problems are due to the non-specific delivery of drugs, the low half-life of drugs, renal clearance, and / or the loss of an antigen on the surface of cancer cells to which the therapeutic molecule is targeted. In addition, for some therapeutic targets, there is no inhibitor approved for clinical use, or available inhibitors are toxic, have short half-lives, or have off-target effects. Therefore, an efficient and specific delivery method of therapeutic molecules to target cells is urgently needed.SUMMARY

[0004] In view of the foregoing, there is a need for systems and methods for efficiently and specifically delivering therapeutic molecules to target cells such as B cells and CD22-positive cells, tissues, and organs, such as blood, lymph nodes, and spleen. This disclosure is directedAttorney Docket No: LLU 24-009 (105781.0238.0) generally to systems, compositions, and methods to address these shortcomings of the art and provide other additional or alternative advantages.

[0005] In one aspect of the present disclosure, a composition comprising a complex comprising a polynucleotide of interest and a carrier is provided. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand.

[0006] In some embodiments, the carrier is a polymer or a lipid. In some embodiments, the carrier is a cationic polymer. In some embodiments, the polymer is polyethyleneimine (PEI), poly- 1-lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), or combination of any thereof, or any chemical or biological modifications of any of these.

[0007] In some embodiments, the carrier is a protein molecule such as a histone or histone- like protein or other polynucleotide binding protein in its natural or biologically modified forms.

[0008] In some embodiments, the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation. In some embodiments, the complex is biotinylated. In some embodiments, the complex is biotinylated, and is bound to streptavidin bound to the CD22 receptor ligand. In some embodiments, the complex comprises N-hydroxy succinimide (NHS)-PEG-biotin. In some embodiments, the carrier and the CD22-receptor ligand are covalently conjugated using homo- or hetero-bifunctional crosslinkers, for example, sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) or 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP).Attorney Docket No: LLU 24-009 (105781.0238.0)

[0009] In some embodiments, the complex is bound to the CD22 receptor ligand via thiolpyridyl thiol conjugation. In some embodiments, the polymer is thiolated and the CD22 receptor ligand is modified to comprise pyridyl thiol.

[0010] In some embodiments, the carrier and the CD22-receptor ligand are synthesized together within bacteria or mammalian host cells, for example, poly-l-lysine fused CD22- receptor’s protein ligand such as antibody, single chain variable fragments (scFv), or anti-CD22 peptides.

[0011] In some embodiments, the carrier and the CD22-receptor ligand are synthesized together within bacteria or mammalian host cells, for example, histone fused CD22-receptor’s protein ligand such as antibody, single chain variable fragments (scFv), or anti-CD22 peptides.

[0012] In some embodiments, the CD22 receptor ligand is an anti-CD22 peptide originating from phage-display library systems.

[0013] In some embodiments, the CD22 receptor ligand is an anti-CD22 antibody. In some embodiments, the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.

[0014] In some embodiments, the CD22 receptor ligand is biotinylated and is bound to the streptavidin.

[0015] In some embodiments, the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).Attorney Docket No: LLU 24-009 (105781.0238.0)

[0016] In some embodiments, the polynucleotide of interest comprises an inhibitory nucleotide sequence against DYRK1A and / or BCL2, and / or other oncogenes, and / or apoptotic genes. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for DYRK1A and / or BCL2 shRNA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for over expression of BIM and / or PUMA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0017] In one aspect, a method of delivering a polynucleotide of interest to a CD22 expressing cell, such as a B cell, is provided. The method includes contacting the B cell with a composition comprising a complex comprising the polynucleotide of interest and a carrier. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand.

[0018] In some embodiments, the carrier is a polymer or a lipid. In some embodiments, the carrier is a cationic polymer. In some embodiments, the polymer is polyethyleneimine (PEI), poly- 1-lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0019] In some embodiments, the carrier is a protein, such as a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.

[0020] In some embodiments, the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation. In some embodiments, the complex is biotinylated. In some embodiments, the complex is biotinylated, and is bound to streptavidin bound to the CD22 receptor ligand. In some embodiments, the complex comprises N-hydroxy succinimide (NHS)-PEG-biotin. In some embodiments, the carrier and the CD22-receptor ligand are covalently conjugated using homo- or hetero-bifunctional crosslinkers, for example, sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) or 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP).

[0021] In some embodiments, the complex is bound to the CD22 receptor ligand via thiolpyridyl thiol conjugation. In some embodiments, the polymer is thiolated and the CD22 receptor ligand is modified to comprise pyridyl thiol.

[0022] In some embodiments, the CD22 receptor ligand is an anti-CD22 antibody. In some embodiments, the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.

[0023] In some embodiments, the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).Attorney Docket No: LLU 24-009 (105781.0238.0)

[0024] In some embodiments, the polynucleotide of interest comprises an inhibitory nucleotide against DYRK1A and / or BCL2. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for DYRK1A and / or BCL2 shRNA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for over expression of BIM and / or PUMA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0025] In one aspect, a method of treating a B cell-associated disease in a subject is provided. The method includes administering to the subject a composition comprising a complex comprising a polynucleotide of interest and a carrier, thereby treating the B cell-associated disease in the subject. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand.

[0026] In some embodiments, the carrier is a polymer, a lipid, or a protein. In some embodiments, the carrier is a cationic polymer. In some embodiments, the polymer is polyethyleneimine (PEI), poly-l-lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P- amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof. In some embodiments, the protein is a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0027] In some embodiments, the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation. In some embodiments, the complex is biotinylated. In some embodiments, the complex is biotinylated, and is bound to streptavidin bound to the CD22 receptor ligand. In some embodiments, the complex comprises N-hydroxy succinimide (NHS)-PEG-biotin. In some embodiments, the carrier and the CD22-receptor ligand are covalently conjugated using homo- or hetero-bifunctional crosslinkers, for example, sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) or 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP).

[0028] In some embodiments, the CD22 receptor ligand is an anti-CD22 antibody. In some embodiments, the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.

[0029] In some embodiments, the CD22 receptor ligand is an anti-CD22 antibody. In some embodiments, the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.

[0030] In some embodiments, the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).

[0031] In some embodiments, the polynucleotide of interest comprises an inhibitory nucleotide against DYRK1A and / or BCL2. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for DYRK1A and / or BCL2 shRNA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expressionAttorney Docket No: LLU 24-009 (105781.0238.0) cassette for over expression of BIM and / or PUMA. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6. In some embodiments, the polynucleotide of interest comprises the pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0032] In some embodiments, the B-cell associated disease is acute lymphoblastic leukemia, such as KMT2A-R. B-ALL or Ph-like B-ALL.

[0033] In one aspect, provided herein is a method of preparing a CD22-targeted polyplex composition. The method includes contacting a polynucleotide with one or more polymers to form a complex; and binding the complex with a CD 22 receptor ligand.

[0034] In some embodiments, the one or more polymers are polyethyleneimine (PEI), poly-1- lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.

[0035] In some embodiments, the one or more polymers are 25 kDa PEI and PBAE447.

[0036] In some embodiments, the method includes binding the complex with the CD22 receptor ligand via streptavidin-biotin conjugation.

[0037] In some embodiments, the method includes binding the complex with the CD22 receptor ligand via thiol-pyridyl thiol conjugation.Attorney Docket No: LLU 24-009 (105781.0238.0)BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. To facilitate this description, like reference numerals designate like structural elements or procedures in a method. Embodiments are illustrated by way of example and not by way of limitation in the figures of the accompanying drawings.

[0040] FIG. 1A schematically depicts mechanism of transgene expression by delivering an anti-CD22 antibody functionalized polyplexes into CD221cells and transgene expression.

[0041] FIG. IB schematically depicts mechanism of treating CD22+cancer using anti-CD22 antibody functionalized polyplexes loaded with an siRNA agent against the oncogene.

[0042] FIG. 2 schematically depicts shRNA-mediated DYRK1 A and BCL2 knockdown and subsequent ALL cell death.

[0043] FIG. 3A schematically depicts polyplex functionalization using biotinylated polyplexes and streptavidin-conjugated antibody. Nanoparticles are prepared using biotinylated polymer and nucleic acid complexes (polyplex) and later coating the surface with streptavidin- conjugated antibodies.

[0044] FIG. 3B schematically depicts siRNA and PEI-based polyplex and maximum length assuming complete linear confirmation.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0045] FTG. 4 depicts the surface expression of CD22 in patient samples (Lax 7 and Lax7R) and KMT2A-r B-ALL cell lines KOPNB, RS4;11, HB11;19, and SEM.

[0046] FIG. 5A depicts surface-bound and internalized Alexa Fluor 488 (AF488, green)- labeled streptavi din-conjugated iron-oxide nanoparticles (SIOPs) of size 150 nm in ALL cell line SEM via the CD22 receptor. FIG. 5B depicts internalization of AF488-labeled SIOPs of size 150 nm by SEM cells via the CD22 receptor. Trypan blue is a known quencher of extracellular green fluorescence. Trypan blue was used to measure the uptake signal as it is known to quench the extracellular green fluorescence of viable cells.

[0047] FIGs. 6A and 6B depict size vs polydispersity index (PDI) of polyplexes (PP) coated with streptavidin and respective antibodies. FIG. 6C depicts net surface charge of PP coated with streptavidin and respective antibodies. Numbers on the X-axis in FIG. 6A indicate the molar ratios of biotinylated polyethyleneimine (PEI) to streptavidin to biotinylated IgG. “PEI” indicates unmodified PEI-based polyplexes.

[0048] FIG. 7 depicts reduction of non-specific association (blue) vs uptake (red) of AF488 labeled polymer-based nucleic acid nanoparticles (PNPs), where the polymer is PEI, with the B cell membrane upon surface coating of PNPs with streptavidin.

[0049] FIG. 8 depicts developing the chemical biotinylation of TgG antibodies with NPB using a biotin quantification kit.

[0050] FIG. 9A depicts total association of AF488-labeled streptavidin as the sole cargo via CD22 by KMT2A-r cell lines, SEM (blue) & HB11;19 (red) cells. FIG. 9B depicts uptake ofAttorney Docket No: LLU 24-009 (105781.0238.0)AF488-labeled streptavidin as the sole cargo via CD22 by KMT2A-r cell lines, SEM (blue) & HBl l;19 (red) cells.

[0051] FIGs. 10A and 10B depict flow cytometry analysis (FIG. 10B) and mean fluorescence intensity (FIG. 10A) of tracrRNA-ATTO488:crRNA polyplex delivery into SEM cells after 48 h. For total association, SEM cells were resuspended in 1X PBS. For internalization, SEM cells were resuspended in 0.4% Trypan Blue. According to the green window analysis, the internalization rate was approximately 100% of the total associated polyplexes. Two-way ANOVA with multiple comparisons; **** indicates P < 0.0001.

[0052] FIGs. 11 A and 1 IB depict flow cytometry analysis (FIG. 11B) and mean fluorescence intensity (FIG. 11A) of tracrRNA-ATTO488:crRNA polyplex delivery into SEM cells after 24 h. For total association, SEM cells were resuspended in IX PBS. For internalization, SEM cells were resuspended in 0.4% Trypan Blue. According to the green window analysis, the internalization rate was approximately 11.5% of the total associated polyplexes. Two-way ANOVA with multiple comparisons; **** indicates P < 0.0001.

[0053] FIG. 12A and FIG. 12B depict size (FIG. 12A) and zeta potential (FIG. 12B) of IgG- functionalized polyplexes in suspension media that resemble blood. Polyplexes of guide RNA (a duplex of tracrRNA and crRNA) were prepared using biotinylated PEI (25 kDa, w / w 0.5) conjugated to streptavidin and PBAE 447 (w / w 2.0). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0054] FIG. 13A and FIG. 13B depict comparison of size characteristics of IgG- functionalized polyplexes in water (FIG. 13B) versus RPMI (FIG. 13A) complete media. Polyplexes of siRNA were prepared using biotinylated PEI (25 kDa, w / w 0.5), streptavidin, andAttorney Docket No: LLU 24-009 (105781.0238.0)PBAE 447 (w / w 2.0). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0055] FIG. 14A and FIG. 14B depict plasmid length and particle size of IgG-functionalized polyplexes in water. Polyplexes of plasmid DNA were prepared using biotinylated PEI (25 kDa, w / w 0.5) conjugated to streptavidin (SV) and PBAE 447 (w / w 2.0). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0056] FIG. 15A and FIG. 15B depict size (FIG. 15A) and charge (FIG. 15B) characteristics of siRNA polyplexes prepared in water, acetic acid, and sodium acetate buffer using biotinylated PET (25 kDa, w / w 0.5) conjugated to streptavidin (SV) and PBAE 447 (w / w 2.0). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0057] FIG. 16 depicts size characteristics of plasmid DNA polyplexes prepared in water, acetic acid, and sodium acetate buffer using biotinylated PEI (25 kDa, w / w 0.5), streptavidin, and PBAE 447 (w / w 2.0). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0058] FIG. 17A and FIG. 17B depict comparison of IgG-functionalized and nonfunctionalized polyplexes in blood-mimicking media. Polyplexes contain guide RNA (a duplex of tracrRNA and crRNA) or plasmid DNA (pUC19) were prepared using biotinylated PEI (25 kDa, w / w 0.5) conjugated to streptavidin and PBAE 447 (w / w of 2.0 for gRNA and 1.0 for pUC19). w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0059] FIGs. 18A-18D depict size and charge characteristics of polyplexes prepared in acetic acid. Polyplexes of siRNA were prepared using biotinylated 25kDa PEI (w / w of 0.5) conjugatedAttorney Docket No: LLU 24-009 (105781.0238.0) to Streptavidin (SV) and PB AE 447 (w / w - variable), w / w was calculated as the weight of polymer over the weight of nucleic acid. FIG. 18A depicts MFI of the fluorescent DNA stain. FIG. 18B depicts zeta potential. FIG. 18C depicts size distribution. FIG. 18D depicts zeta potential distribution.

[0060] FIGs. 19A-19C depict size and charge characteristics of polyplexes prepared in acetic acid. Polyplexes of siRNA were prepared using biotinylated 25kDa PEI (w / w of 0.5) conjugated to Streptavidin (SV) and PBAE 447 (w / w - variable), w / w was calculated as the weight of polymer over the weight of nucleic acid. FIG. 19A depicts MFI of the fluorescent DNA stain. FIG. 19B depicts size distribution. FIG. 19C depicts zeta potential distribution.

[0061] FIGs. 20A-20C depict size and charge characteristics of polyplexes prepared in acetic acid. Polyplexes of siRNA were prepared using biotinylated 25kDa PEI (w / w of 0.5) conjugated to Streptavidin (SV) and Protamine sulfate (PS, w / w - 1.0 or 2.5). w / w was calculated as the weight of polymer over the weight of nucleic acid. FIG. 20A depicts size distribution. FIG. 20B depicts size characteristics. FIG. 20C depicts zeta potential.

[0062] FIGs. 21A and 21B depict gel electrophoresis (FIG. 21A) and signal quantitation (FIG. 21B) showing condensation of siRNA polyplexes with increasing protamine sulfate ratios and combinations with PEI-streptavidin complexes. Polyplexes of siRNA were prepared using biotinylated 25kDa PEI (w / w of 0.5) conjugated to Streptavidin (SV) and Protamine sulfate (PS, w / w - variable), w / w was calculated as the weight of polymer over the weight of nucleic acid.

[0063] FIGs. 22A and 22B depict gel electrophoresis (FIG. 22A) and signal quantitation (FIG. 22B) showing condensation characteristics of siRNA polyplexes prepared in acetic acid using 25 kDa PEI (P25) (w / w of 2.0). / w was calculated as the weight of polymer over the weightAttorney Docket No: LLU 24-009 (105781.0238.0) of nucleic acid. Gel electrophoresis shows progressive condensation of siRNA with increasing PBAE 447 ratios (w / w 20-50). Ladder bands indicate size markers for nucleic acid migration.

[0064] FIGs. 23A-23D depict size and charge characteristics of polyplexes prepared in acetic acid. Polyplexes of plasmid DNA (pUC19) were prepared in acetic acid using 25 kDa PEI (P25) (w / w 2.0) or PBAE 447 (w / w - variable), w / w is calculated as the weight of polymer over the weight of nucleic acid. FIG. 23A depicts size distribution and zeta potential, with particle size reduction with increasing PBAE 447 ratios. FIG. 23B depicts gel electrophoresis. FIG. 23C depicts size distribution. FIG. 23D depicts relationship of size and volume.

[0065] FIG. 24 depicts flow cytometry analysis of CD22 surface expression in B-ALL cell lines and patient samples. Blue = unstained; Red = stained with mouse anti-human CD22 antibody followed by FITC-conjugated goat anti-mouse secondary antibody (Fab2). Cell lines in green are KMT2A-R B-ALL and in purple are Ph-like B-ALL. Ramos is a Burkitt’s Lymphoma Cell line.

[0066] FIGs. 25A-25C depict delivery of tracrRNA-ATTO488 polyplexes into SEM-Cas9 cells using CD22 antibody-functionalized polyplexes. tracrRNA-ATTO 488 polyplexes were prepared using P25-SV (biotinylated 25 kDa PEI conjugated to streptavidin) at a polymermucleic acid weight ratio (w / w) of 2.0 and functionalized with biotinylated IgG, biotinylated anti-CD22 antibody (aCD22), or biotinylated BSA at a ratio of 1 :0.1. Samples were incubated with SEM- cas9-puro cells in serum-free RPMI for 4 hours, followed by RPMI complete medium. Association and internalization were quantified by flow cytometry under varying IgG / aCD22 ratios. FIG. 25A depicts cell viability and tracrRNA positive cell ratio. FIG. 25B depicts association and internalization. FIG. 25C depicts comparison of total association and internalization of tracrRNA- ATTO488 polyplexes functionalized with varying ratios of IgG or anti-CD22 antibody relative toAttorney Docket No: LLU 24-009 (105781.0238.0)BSA (100:0, 75:25, 50:50, 25:75, and 0:100). Data demonstrate enhanced uptake and internalization with increasing anti-CD22 antibody content, confirming receptor-mediated delivery via CD22.

[0067] FIGs. 26A and 26B depict size (FIG. 26A) and charge (FIG. 26B) characteristics of IgG-functionalized polyplexes prepared in water. Polyplexes of siRNA were prepared using thiolated (-SH) 25kDa PEI (w / w of 2.0, thiol generated using sulfo-LC-SPDP) conjugated to IgG (at PELIgG = 1 :0.1) via pyridyl thiol (PT) generated using sulfo-LC-SPDP. TCEP and PABA (4- ABA) were used to control the reaction between -SH and PT. w / w is calculated as the weight of polymer over the weight of nucleic acid.

[0068] FIGs. 27A-27C depict size and charge characteristics of IgG-functionalized polyplexes prepared in water and resuspended in water or RPMI complete media. Polyplexes of single guide RNA (sgRNA) were prepared using unmodified 25kDa PEI (w / w of 1.0) and biotinylated 25kDa PEI (w / w of 1.0) conjugated Streptavidin and to biotinylated IgG (at PEI: IgG = 1 :0.1). w / w is calculated as the weight of polymer over the weight of nucleic acid. FIG. 27A depicts size and charge characteristics. FIG. 27B depicts size distribution. FIG. 27C depicts zeta potential distribution.

[0069] FIGs. 28A-28C depict size and charge characteristics of IgG-functionalized polyplexes prepared in water. Polyplexes were made using mRNA and 1 / 10 parts of P25-IgG or P25-BSA and 9 / 10 parts of unmodified P25. P25-IgG or P25-BSA were achieved by using thiolated (-SH) 25kDa PEI (thiol generated using sulfo-LC-SPDP) conjugated to IgG or BSA (at PETIgG or PEI:BSA = 1:0.1) via pyridyl thiol (PT) generated using sulfo-LC-SPDP. P25 is 25kDa branched PEI. 2 -ME (b-ME) was used to check the aggregation of polyplexes during DLS. FIG. 28A depictsAttorney Docket No: LLU 24-009 (105781.0238.0) size distribution of non-reduced polyplexes. FIG. 28B depicts size distribution of b-ME treated polyplexes. FIG. 28C depicts zeta potential.

[0070] FIGs. 29A-29F depict size and charge characteristics of IgG-functionalized polyplexes prepared in water. Polyplexes were made using mRNA and P25-IgG or P25-BSA. P25-IgG or P25-BSA were achieved by using thiolated (-SH) 25kDa PEI (thiol generated using sulfo-LC- SPDP) conjugated to IgG orBSA (atPEI:IgG orPEI:BSA = 1 :0.1) via pyridyl thiol (PT) generated using sulfo-LC-SPDP. P25 is 25kDa branched PEI. 2-ME (b-ME) was used to check the aggregation of polyplexes during DLS. FIGs. 29A-29C depict size distribution of mRNA polyplexes functionalized with thiolated PEI (FIG. 29A), IgG and thiolated PEI (FIG. 29B), and BSA and thiolated PEI (FIG. 29C), respectively. FIG. 29D depicts zeta potential. FIGs. 29E-29F depict size and charge characteristics of non-reduced (FIG. 29E) or reduced (FIG. 29F) polyplexes.

[0071] FIGs. 30A-30D depict Size and charge characteristics of IgG-functionalized polyplexes prepared in water and resuspended in water. Polyplexes of single guide RNA (KMT2A sgRNA) or mRNA (GFP mRNA) or mRNA plus sgRNA (msgRNA) were prepared using biotinylated 25kDa PEI (w / w of 2.0) conjugated Streptavidin and to biotinylated IgG (at PEI: IgG = 1 :0.1). w / w is calculated as the weight of polymer over the weight of nucleic acid. FIG. 30A depicts size distribution. FIG. 30B depicts volume distribution. FIG. 30C depicts size characteristics. FIG. 30D depicts zeta potential.

[0072] FIG. 31 depicts size and charge analysis of CRLF2 sgRNA polyplexes prepared using biotinylated PEI and streptavidin-IgG conjugation. Larger particle sizes were observed, likely dueAttorney Docket No: LLU 24-009 (105781.0238.0) to secondary structure in sgRNA. sgRNA is used in CRISPR / Cas9 mediated deletion of target genes (such as CRLF2) in host cells.

[0073] FIGs. 32A and 32B depict effect of anti-DYRKlA targeting siRNA polyplexes in SEM cells. Cells and formulation were suspended in RPMI complete media.

[0074] 0.5 fmol / cell siRNA (FIG. 32A) did not show significant knockdown efficiency compared to control. In contrast, 1 fmol / cell (FIG. 32B) showed efficient knockdown. BL is biolegend antibody and SB is sinobiological.

[0075] FIGs. 33A and 33B depict effect of ar -DYRKlA targeting siRNA polyplexes in SEM cells. Cells were treated with anti-DYRKl A targeting siRNA polyplexes in 1 ml serum-free RPMI for 4 h followed by addition of 1 ml RPMI complete medium. FIGs. 33A and 33C depict no difference in apoptosis between IgG or anti-CD22 antibody functionalized polyplexes, as measured by flow cytometer. FIGs. 33B and 33D depict knockdown efficiency as measured by quantitative PCR, showing -33% reduction in DYRK1A expression in cells treated with anti- CD22 antibody functionalized anti-DYRKlA targeting siRNA polyplexes relative to IgG- functionalized control.

[0076] FIG. 34A depicts gel retardation assay of siRNA polyplexes prepared with increasing IgG ratios (PEI: IgG from 1 :0.01 to 1 : 1), confirming complete siRNA condensation across all IgG ratios tested. FIG. 34B depicts dynamic light scattering (DLS) analysis of siRNA polyplexes prepared with increasing IgG ratios (PELIgG from 1:0.01 to 1 : 1). siRNA polyplex sizes are smaller (DLS) until a certain quantity of IgG indicating the possibility of successful transfection of siRNA into host cells.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0077] FTG. 35 depicts gel migration assay showing proper condensation of siRNA and plasmid DNA (4.7 kb) using PEI polymers. PEI25 at w / w 0.5 was sufficient for complete condensation.FIGS. 36A-36F DEPICT TESTING OF THE SIRNA CONDENSATION ABILITY OF DIFFERENT POLYMERS (2 KDA PEI (FIG. 36A), 10 KDA PEI (FIG. 36B), 25 KDA PEI (FIG. 36C), 70 KDA PEI (FIG. 36D)) AND PROTAMINE (FIG. 36E) BY THE COMPACTNESS OF SIRNA POLYPLEXES ANALYZED BY SYBR EXCLUSION ASSAY (FIGS. 36A-36E) AND GEL RETARDATION ASSAY (FIG. 36F). BOTH EXPERIMENTS INDICATE EFFICIENT CONDENSATION AND COMPACTNESS OFSIRNA POLYPLEXES AT LOW WEIGHT RATIOS.Attorney Docket No: LLU 24-009 (105781.0238.0)DETAILED DESCRIPTION

[0078] The present disclosure describes various embodiments related to compositions and methods for delivering a molecule of interest (such as a therapeutic molecule, such as a polynucleotide) to target cells such as B cells and CD22-positive cells, tissues, and organs, such as blood, lymph nodes, and spleen B cells, and methods of treating B cell-associated diseases (such as B cell cancer, such as ALL, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia) in subjects using the compositions and methods provided herein. In the following description, numerous details are set forth in order to provide a thorough understanding of the various embodiments. Before the present methods and compositions are described, it is to be understood that these embodiments are not limited to particular methods or compositions described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present embodiments will be limited only by the appended claims. The description may use the phrases “in certain embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous.I. Definitions

[0079] A “subject” refers an animal, such as a mammal, including a primate (such as a human, a non-human primate, such as a monkey) and a non-primate (such as a mouse). In some aspects ofAttorney Docket No: LLU 24-009 (105781.0238.0) the disclosure, the subject is a human. In some aspects, the subject is a pediatric subject, such as a neonate, an infant, or a child. In other aspects, the subject is an adult subject.

[0080] A “patient” refers to a subject who shows symptoms and / or signs of a disease, is under treatment for disease, has been diagnosed with a disease, and / or is at risk of developing a disease. A “patient” can be human and veterinary subjects. Any reference to subjects in the present disclosure, should be understood to include the possibility that the subject is a “patient” unless clearly dictated otherwise by context. More specifically, the subject in certain aspects is a patient who has a B cell-associated disease, such as ALL.

[0081] As used herein, the terms “treating”, “treatment” and the like, shall include the management and care of a subject or patient for the purpose of combating a disease, condition, or disorder and includes the administration of a composition to prevent the onset of the symptoms or complications, alleviate the symptoms or complications, reduce at least one associated sign, symptom, or condition, or eliminate the disease, condition, or disorder. Treatment also refers to a prophylactic treatment, such as prevention of a disease (such as ALL) or prevention of at least one sign, symptom, or condition associated with the disease. Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0082] An “N / P ratio” as used herein refers to the ratio of positively charged amine groups (N) of the carrier (such as the polymer) and negatively charged phosphate groups (P) of the polynucleotide of interest. Nucleic acid cargos are loaded to cationic carriers via the electrostatic interaction between positively charged amine groups (N) of the carrier and negatively charged phosphate groups (P) of RNA or DNA. The N / P ratio determines the loading / complexing / condensing efficiency of the cationic polymer carrier.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0083] An “antibody” refers to a peptide or polypeptide derived from, modeled after, or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. An antibody includes an immunoglobulin molecule having two heavy chains and two light chains prepared by any method known in the art or later developed. Antibodies can be produced by immunizing mice, rats, or rabbits or by genetic engineering methods such as cloning of native immunoglobulin genes (or humanized immunoglobulin genes) in mammalian plasmid vectors and then expressing them in mammalian cell lines. An antibody also refers to and includes an antibody fragment. An “antibody fragment” refers to a fragment of an antibody that retains capacity to bind an antigen, and includes an antigen binding site (e g., a fragment, a subsequence, a complementarity determining region (CDR)); a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab’)2 fragment or a Fab2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment, which consists of a VH domain; and an isolated complementarity determining region (CDR). For example, a fragment of an antibody molecule, produced by chemical cleavage or genetic engineering techniques, is an antibody. A single chain variable fragment (scFv), which is a fusion protein of the variable regions of the heavy and light chains (VH and VL) of immunoglobulins connected with a short linker peptide of 10-25 amino acids, such as that produced using combinatorial genetic libraries and phage display technologies, is an antibody.

[0084] As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” refers to a detectable (such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,Attorney Docket No: LLU 24-009 (105781.0238.0)80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) negative change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass both a partial reduction and a complete reduction compared to a control.

[0085] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” or “enhanced” or “enhancing” or “enhance” refers to a detectable ( such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%) positive change in the parameter from a comparison control, such as an established normal or reference level of the parameter, or an established standard control.II. Compositions for Delivery of Cargo via CD22

[0086] Provided herein is a composition comprising a complex comprising a polynucleotide of interest and a carrier. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand. Any CD22 receptor ligand that binds the complex to the CD22 receptor can be used, including an anti-CD22 antibody and a glycan ligand. CD22 is a B-cell-specific receptor known to shuttle cargo from outside to inside B cells. CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family that is known to be a regulator of B cell signaling. Its B cell-specific expression makes it an attractive target for immunotoxin-mediated B cell depletion therapy for the treatment of B cell lymphomas and autoimmune diseases. Although CD22 is well documented to be an endocytic receptor, it is believed that following internalization it is targeted for degradation. However, CD22 is instead constitutively recycled to the cell surface. Glycan ligand-based cargo is released from CD22 and accumulates intracellularly as CD22 recycles between the cell surfaceAttorney Docket No: LLU 24-009 (105781.0238.0) and endosomal compartments. Tn contrast, antibodies to CD22 do not accumulate, but remain bound to CD22 and recycle to the cell surface. This mechanism enables development of agents provided herein that target CD22 as an endocytic receptor for delivery of cytotoxic cargo to B cells.

[0087] The carrier can be a polymer or a lipid, forming a polyplex or a lipoplex, respectively, with the polynucleotide of interest. The carrier is a cationic polymer. A polymer and the polynucleotide of interest can form a polymer-based nucleic acid nanoparticle (PNP). Such a complex can be referred to as a polyplex.

[0088] For example, the polymer can be polyethyleneimine (PEI), poly-l-lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.

[0089] The carrier can also be a protein, for example a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.

[0090] The complex (for example, a PNP) can be biotinylated, and can be bound to streptavidin bound to the CD22 receptor ligand. The complex can contain N-hydroxysuccinimide (NHS)-PEG-biotin. The CD22 receptor ligand can be biotinylated and can be bound to the streptavidin. An example complex is depicted in FIG. 3.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0091] Alternatively or additionally, the complex can be bound to the CD22 receptor ligand via thiol-pyridyl thiol conjugation. For example, the polymer can be thiolated (-SH) and the CD22 receptor ligand can be modified to comprise pyridyl thiol (PT). -SH or PT can be introduced using available methods, for example, using sulfo-LC-SPDP. TCEP and PABA (4-ABA) can be used to control the reaction between -SH and PT.

[0092] The carrier and the CD22-receptor ligand can be covalently conjugated using homo- or hetero-bifunctional crosslinkers, for example, sulfosuccinimidyl 6-(3'-(2- pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) or 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP).

[0093] The CD22 receptor ligand can be any chemical compound or a macromolecule that can attach to CD22 on the cell surface and aid in the delivery of the nucleic acid cargo to the CD22- expressing cells. For example, the CD22 receptor ligand can be an anti-CD22 antibody. The anti- CD22 antibody can be an anti-CD22 single-chain variable fragment (scFv) antibody.

[0094] The carrier and the CD22-receptor ligand can be synthesized together within bacteria or mammalian host cells, for example, poly-l-lysine fused CD22-receptor’ s protein ligand such as antibody, single chain variable fragments (scFv), or anti-CD22 peptides.

[0095] The carrier and the CD22-receptor ligand can be synthesized together within bacteria or mammalian host cells, for example, histone fused CD22-receptor’s protein ligand such as antibody, single chain variable fragments (scFv), or anti-CD22 peptides.

[0096] The CD22 receptor ligand can be an anti-CD22 peptide originating from phage-display library systems.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0097] The polynucleotide of interest can contain any type or size of polynucleotide, including one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).

[0098] The polynucleotide of interest can include an inhibitory nucleotide against DYRK1A and / or BCL2. For example, the polynucleotide of interest contains pDNA comprising an expression cassette for DYRK1A and / or BCL2 shRNA. pDNA can be used for stable transformation of target cells (such as B cells) for a long-term (or perpetual) effect.

[0099] The polynucleotide of interest can also include a pDNA comprising an expression cassette for over expression of BIM and / or PUMA; a pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or a pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0100] The formulation of the complexes (for example polyplexes) provided herein can be adjusted as shown in the Examples. Any combinations and formulations of complexes provided in the Examples provided herein are contemplated in the compositions and methods provided herein.

[0101] Also provided herein is a method of preparing a CD22-targeted composition. The method includes contacting a polynucleotide with a carrier, such as one or more polymers to form a complex; and binding the complex with a CD 22 receptor ligand.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0102] In some embodiments, the one or more polymers are polyethyleneimine (PEI), poly-1- lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.

[0103] In some embodiments, the one or more polymers are 25 kDa PEI and PBAE447.

[0104] In some embodiments, the method includes binding the complex with the CD22 receptor ligand via streptavidin-biotin conjugation. In some embodiments, the method includes binding the complex with the CD22 receptor ligand via thiol-pyridyl thiol conjugation. Any compositions provided herein can be used and combined at any ratio to produce a complex suitable for CD22-targeted delivery of cargo of interest to CD22 positive cells.III. Delivery of Polynucleotides to B Cells via CD22

[0105] Provided are methods of delivering therapeutic cargo, such as a polynucleotide of interest, to a CD22 expressing cell, such as a B cell. The method can deliver therapeutic cargo to any CD22 expressing cell or tissue, including B cells, blood, lymph nodes, and spleen. The method includes contacting the cell (such as the B cell) with the composition provided herein. The methods of delivering therapeutic cargo provided herein entail the use of CD22 surface receptor-mediated endocytosis to deliver polynucleotides of interest (polynucleotide of any suitable type or size, including linear dsDNA, linear ssDNA, pDNA, mRNA, siRNA, shRNA, mRNA, tRNA, CRISPR cas9 or cas 12-based gRNA, oligonucleotides, or base modified DNA or RNA) to CD22-positive cells that are of human origin or otherwise. The surface of nucleic acid complexes (such as lipoplexes or polyplexes prepared using lipids or polymers, respectively) is coated with a ligandAttorney Docket No: LLU 24-009 (105781.0238.0) of the CD22 receptor. This ligand could be any chemical compound or a macromolecule that can attach to CD22 on the cell surface and aid in the delivery of the nucleic acid cargo to the CD22- expressing cells.

[0106] For example, the method includes contacting the B cell with a composition comprising a complex comprising the polynucleotide of interest and a carrier. The surface of the complex is coated covalently or non-covalently with a CD22 receptor ligand. For example, the methods provided herein can deliver plasmid DNAs (pDNAs) that constitutively express therapeutic molecules (for example, shRNAs, such as anti-DYRKlA and anti-BCL2 shRNAs) specifically to ALL cells in vivo (FIG. 2) to avoid the toxic off-target effects that pharmacologic inhibitors (for example, DYRK1A or BCL2 inhibitors) may have.

[0107] A polynucleotide of interest can also include a pDNA comprising an expression cassette for over expression of BIM and / or PUMA; a pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or a pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0108] For example, with CD22 as a therapeutic target on cell surface (such as B cell surface), the polynucleotides and polycationic polymer complexes (polyplexes) can be functionalized with a CD22 antibody or scFv fragment. With the internalization of polynucleotides via the CD22 receptor, the polynucleotides can be delivered and exert their therapeutic effect in the cells.

[0109] The expression of CD22 on the cell surface of ALL cells has been validated (FIG. 4). The polynucleotide delivery system provided herein can comprise polymer-nucleic acidAttorney Docket No: LLU 24-009 (105781.0238.0) complexes (nucleic polyplexes) of consistent size (about 120-150nm) and polydispersity index (PDI, about 0.2) which can be taken up and internalized by CD22 receptor.

[0110] The CD22-targeing polynucleotide delivery system provided herein can be used in a novel PEI-based cationic polymer complex treatment strategy using CD22 on the surface of B- ALL cells to transport DYRK1 A shRNA to the nucleus to inhibit DYRK1A translation. Unlike signaling inhibitors or CAR-T cell therapies, the PEI-based nucleic acid delivery strategy has no off-target effects and can be generated easily and cost-effectively, and can be made universally available to any patients with B-ALL. The system can include PEI-based polymer complexes containing psADYRKlA (polyplexes) and conjugated with antibodies against CD22. The binding of the p.s / rDYRK l A-containing polymer complex to CD22 induces rapid internalization of CD22 and consequently delivers the cargo to the endosome. The p.s / vDYRK l A that escapes the endosome translocates to the nucleus where shRNA is expressed (FIG. 1). Later, the shRNA that translocates to the cytoplasm helps the DICER / RISC complexes to degrade DYRK1 A mRNA consequently inducing apoptosis in ALL cells (FIG. 2).

[0111] The CD22 receptor targeting polymer-based nucleic acid delivery system can include the cationic polymer, 25 kDa PEI, which is used effectively in gene delivery systems. PEI can be endowed with exclusive transfection abilities by simply conjugating it to a cell-specific receptor’s ligand of interest. In the CD22-targeing delivery system provided herein, the PELplasmid complexes can be functionalized with anti-human CD22 scFv fragments using Streptavidin as an adapter molecule (FIG. 3). This non-covalent bioconjugation strategy can be readily applied to different ligand and receptor pairs for any cell of interest.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0112] The CD22-targeing delivery system provided herein can include nanosized cationic polymer-based nanosized complexes (polymer nanoparticles, PNPs) containing shRNA- expressing plasmids and conjugated with antibodies against CD22. Plasmid DNA and polymer complexes (polyplexes) prepared in pure water can have a hydrodynamic diameter of about 20- 200 nm, such as about 20-50 nm, 50-75 nm, 75-100 nm, 100-125 nm, 125-150 nm, 150-175 nm, 175-200 nm, 75-200 nm, or about 20 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, or 200 nm. CD22 receptor is known to be internalized into B cells upon antibody attachment. CD22 follows the clathrin-mediated endocytosis (CME) route of internalization which was reported to have vesicle sizes of 150-200 nm. The coat protein, clathrin, can coat vesicles of diametrical size 150-200 nm. Cargo of less than 200 nm attached to CD22 antibodies may effectively be internalized into B cell-derived acute lymphoblastic leukemia (B-ALL) cells. The binding of the plasmid-containing cationic polymer complex to CD22 can induce rapid internalization of CD22 and consequently can deliver the cargo to the endosome, where the PEl-based nucleic acid complex can escape degradation followed by the expression of transgene CD22 is known to be recycled back to the cell surface in the absence of intervening factors. However, the presence of polymers such as PEI are known to rupture the endolysosome due to the so-called “proton-sponge” effect. The escaped polyplex can be translocated into the nucleus where the expression of transgenes occurs. (FIG. 1)

[0113] For example, after the B-ALL cells internalize the cargo via CD22, the polynucleotide of interest is translocated into the nucleus. In specific embodiments, the polynucleotide of interest is an pDNA, which then constitutively expresses therapeutic molecules, such as anti- DYRK1A and anti-BCL2 shRNA, in the nucleus. The synthesized shRNAs are then translocated into the cytosol where the DICER complex cuts the shRNAs to generate siRNAs. siRNAs are then used asAttorney Docket No: LLU 24-009 (105781.0238.0) a guide by the RISC complex which cleaves specific mRNAs. Due to this translational silencing of the targets, DYRK1 A and BCL2, BIM-mediated apoptosis will occur in B-ALL cells (FIG. 2).

[0114] The polynucleotide of interest can also include a pDNA comprising an expression cassette for over expression of BIM and / or PUMA; a pDNA comprising an expression cassette for shRNA against oncogenes such as RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or a pDNA comprising an expression cassette for shRNA against the upstream or downstream pathway gene targets of oncogenes.

[0115] The advantages of the CD22-targeing delivery system provided herein include the following. The costs of manufacturing this formulation are less than generating CAR-T cells in adoptive T cell therapy. Not only well-equipped research centers but also small hospitals with minimal equipment can prepare the formulation provided herein in a few hours. In addition, unlike the CAR-T system that requires evaluations such as regulating the activation profile to prevent CAR-T cells from undergoing exhaustion or activation-induced cell death, the CD22-targeing delivery system provided herein does not need evaluations. Importantly, side effects like cytokine release syndrome, which is common during CAR-T cell therapy, are not expected in the therapy using the CD22-targeing delivery system provided herein. Finally, the CD22-targeing delivery system provided herein can be modified to load and deliver therapeutic molecules targeting a molecule of interest, to treat a variety of diseases including ALL, AML, CLL, and, solid tumors.IV. Treating B Cell-Associated Disease

[0116] A method of treating a B cell-associated disease in a subject is provided. The method includes administering to the subject a composition comprising a complex comprising a therapeutic molecule (such as a polynucleotide of interest) and a carrier, thereby treating the BAttorney Docket No: LLU 24-009 (105781.0238.0) cell -associated disease in the subject. The surface of the complex is coated covalently or non- covalently with a CD22 receptor ligand.

[0117] Polynucleotides can have long-lasting effects or transient effects on the cell. For example, pDNA once entered into the cells can constitutively express a gene of interest (such as an apoptotic gene) until the cancer cell dies. pDNA can also express a vital protein that is not expressed in a diseased B cell. mRNAs can be used to express foreign genes until the life of that mRNA molecule. siRNAs can be used to silence an oncogenic gene or the expression of a diseasecausing protein. The nucleic acid delivery strategy via the CD22 receptor is cell-specific and prevents patients from going into relapse conditions of cancers. The CD22-mediated polynucleotide delivery provided herein can be readily applied to deliver a therapeutic molecule to B cells, for example to treat B cell associated disease or condition, such as B-cell cancer (for example ALL, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B- cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia).

[0118] B cell associated diseases for which the CD22-targeing delivery system provided herein can be used and which can be treated by the methods provided herein include ALL. ALL is a prevalent cancer in both children and adults, with children and adolescents alone accounting for about 50% of all cases. In particular, the presence of KMT2A (also known as mixed lineage leukemia, or MLL) rearrangement in ALL is an independent dismal prognostic factor with longterm survival rates of less than 60% across all age groups. Rearranged KMT2A (KMT2A-r) ALL presents a complex clinical challenge, with a high incidence in infants and a tendency for aggressive relapse. The KMT2A gene (formerly MLL1 / MLL / ALL-1 / HRX / HTRX1) belongs toAttorney Docket No: LLU 24-009 (105781.0238.0) the group of KMT genes that catalyze the transfer of methyl groups from S-adenosylmethionine to the lysine residues on histone tails, especially H3, and is one of the most promiscuous recombination hot spots of the human genome with regard to the onset of malignant diseases.

[0119] The methods provided herein can be used to treat B cell-associated disease using the CD22-targeing polynucleotide delivery system provided herein, to deliver polynucleotides of any type or size, including linear dsDNA, linear ssDNA, pDNA, mRNA, siRNA, shRNA, mRNA, tRNA, CRISPR cas9 or cast 2-based gRNA, oligonucleotides, or base modified DNA or RNA. For example, the methods provided herein can treat ALL by administering to the subject the composition provided herein containing inhibitory polynucleotides (such as shRNA and siRNA) against DYRK1A and / or BCL2 for treatment of B-ALL. Dual specific tyrosine regulated kinase 1A (DYRK1A) is one of the molecules required for KMT2A-r ALL and / or Ph-like ALL proliferation and survival. The presence of ERK signaling activating mutations (for example FLT3 and RAS pathway mutations) sensitizes ALL cells to DYRK1A inhibition. KMT2A-r ALL cases have concomitant ERK activating mutations (FLT3 -25% and RAS pathway -47%). Pharmacological inhibition of DYRK1A using three different inhibitors (EHT1610, harmine, and GNF2133) significantly inhibited cell proliferation, with the number of cells in S-phase significantly reduced. Mechanistically, DYRK1A inhibition results in the upregulation of pro- apoptotic BIM, which is negatively regulated by BCL2. DYRK1A inhibition synergistically kills RAS-driven ALL cells when given in combination with a BCL2 inhibitor.

[0120] While multiple DYRK1 A inhibitors have been generated and demonstrated promising results in vitro, the clinical application of pharmacological DYRK1A inhibition in vivo is challenging. Currently available DYRK1 A inhibitors have poor pharmacodynamics and toxic off- target effects. For example: l) EHT1610 is expensive and highly toxic in mice, 2) harmine,Attorney Docket No: LLU 24-009 (105781.0238.0) although cheaper to produce is reported to show hallucinogenic effects in humans in clinical testing, and 3) other experimental DYRK1 A inhibitors (e.g., GNF2133) have been generated, but demonstrate poor pharmacodynamics, and induce toxic off-target effects in mice. The CD22- targeing delivery system provided herein can overcome these current limitations by delivering plasmid DNAs (pDNAs) that constitutively express anti-DYRKlA and anti-BCL2 shRNAs to CD22 expressing cells (such as B cells) to avoid the toxic -target effects that the pharmacologic inhibitors have.

[0121] Any types, sizes, and sequences of polynucleotides of interest can be delivered using the CD22-targeing system provided herein. Polynucleotides of interest in the compositions, systems, and methods provided herein include linear dsDNA, linear ssDNA, pDNA, mRNA, siRNA, shRNA, mRNA, tRNA, CRISPR cas9 or casl2-based gRNA, oligonucleotides, or base modified DNA or RNA. In specific embodiments, the methods, systems, and compositions provided herein comprise pDNA, which can constitutively express the exogenous gene for the entire life span of the host cell upon internalization into the cell. The polynucleotide of interest that is delivered using the CD22-targeting system provided herein can include an inhibitory nucleotide against DYRK1 A and / or BCL2, such as a pDNA comprising an expression cassette for DYRK1 A and / or BCL2 shRNA; a pDNA comprising an expression cassette for overexpression of BIM and / or PUMA; a pDNA comprising an expression cassette for shRNA against RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or a pDNA comprising an expression cassette for shRNA against an upstream or downstream pathway gene target of any one of RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6.

[0122] Advantages of the CD22-mediated delivery systems, methods, and compositions provided herein include efficiency of administration: ALL cells need to endocytose the pDNAAttorney Docket No: LLU 24-009 (105781.0238.0) cargo only once for the therapy to be effective, in contrast to antibody-drug conjugate (ADC) therapies where repeatable doses need to be administered. Moreover, the CD 19 CAR-T therapy is known to become ineffective and lead to relapse conditions in ALL patients because the surface antigen (CD 19) on ALL cells is lost during the treatment. The therapeutic pDNA delivery via the CD22-targeing systems provided herein for effective delivery of the therapeutic pDNA to B cells to provide lasting therapeutic effects by a single administration can offer efficiency and improved clinical outcomes relative to the available treatment options.

[0123] According to the methods provided herein, the composition provided herein can be administered to the subj ect at any dose and intervals that are suited for treating the B cell-associated disease, such as ALL. A skilled artisan can identify and practice an appropriate dosing regimen suited for the clinical purpose. For example, the composition comprising pDNA may need to be delivered once, or limited times (such as twice, three times, four times, five times) to treat the B cell-associated disease, as pDNA once entered into the cells may be able to constitutively express a molecule of interest (such as an apoptotic gene) until the cancer cell dies. Additionally or alternatively, the composition may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In some embodiments, the composition is administered at the frequency required to achieve a desired effect.

[0124] The schedule can involve closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6Attorney Docket No: LLU 24-009 (105781.0238.0) hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the composition is not administered. For example, the composition can be initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect.

[0125] The composition can be administered to the subject via any desired route. For example, the composition can be administered to the subject orally, intravenously, subcutaneously, intramuscularly, or intrathecally. Further, any standard methods for treating B cell-associated disease, including chemotherapy, radiation, bone marrow transplant, and stem cell transplant, can be used with (such as sequentially or concurrently) the methods provided herein.EXAMPLES

[0126] The following examples are offered by way of illustration and not by way of limitation.Example 1: Development of the polymer-based nucleic acid delivery system effective for CD22+ cells

[0127] A novel transfection-based nucleic acid delivery system using ligands that specifically identify the CD22 surface receptor on cells, such as ALL cells is developed for a guided knockdown of DYRK1A and BCL2 in CD22+high-risk KMT2AR and Ph-like ALL cells. A pre- clinical treatment strategy is developed that is based on DYRK1A and BCL2 knockdown. Transfection is a process in which exogenous (foreign) polynucleotides are delivered to and expressed in host cells. In contrast to its viral counterparts used in generating CAR-T cells, a polymer, such as polyethyleneimine (PEI) (a non-viral carrier and a widely employed cationic polymer), can complex virtually any size of DNA. Further, PEl’s low-immune recognition allowsAttorney Docket No: LLU 24-009 (105781.0238.0) it to be an effective gene carrier in vivo. Due to the presence of positively charged amines in PEI at physiological pH, nucleic acid condensation is rapidly achieved by masking the negatively charged phosphate groups in its backbone that would otherwise cause them to stretch and relax due to repulsions. Typically, the polymer (nitrogen in amine groups, “N”) and nucleic acid (phosphate, “P”) complexes (polyplexes) are prepared at an N / P ratio of 30 or a weight ratio of 2. Polymer-based nucleic acid nanoparticles (PNPs) that associate with the host cell by charge-charge or ligand-receptor interactions, enter the cell via endocytosis. Subsequently, polynucleotides that otherwise are degraded, are known to escape from the endolysosomes due to the virtue of polymers such as PEI in a so-called “proton-sponge” effect, a phenomenon in which endolysosomes rupture due to osmotic swelling and swelling of PEI due to the repulsive forces between the positively charged amines (FIG. 2). The PNPs are functionalized with conjugates of streptavidin and antibodies against human CD22 (hCD22) (FIG. 3). These functionalized PNPs can specifically interact with CD22 surface receptors on B cells and ALL cells. CD22 is constitutively internalized via clathrin-mediated endocytosis (CME), and thus, can be targeted for drug delivery. The coat protein, clathrin, is observed to be able to coat vesicles of diametrical size 150-200 nm3.

[0128] KMT2A-r ALL cells and patient samples express abundant CD22 surface receptors (FIG. 4). To test whether particles of defined size can be uptaken by CD22+ALL cells, the streptavidin-conjugated iron-oxide nanoparticles (150 nm) were coated with biotinylated CD22 or IgG antibodies. Strikingly, these magnetic nanoparticles of size -150 nm are specifically internalized when conjugated to CD22 antibodies, but not when conjugated to IgG antibodies (FIGs. 5A & 5B). The results may suggest that PNPs that fall within the size range of the CD22- receptor-mediated endocytosis can be effectively delivered to CD22+ALL cells.Addressing PEI’s cytotoxicityAttorney Docket No: LLU 24-009 (105781.0238.0)

[0129] Given PEI’s exclusive transfection abilities, streptavidin can be used as an adapter molecule to conjugate PEI to CD22 antibody or other cell-specific receptor’s ligand of interest (FIG. 3). PEI can interact with anionic cell surfaces. Even free PEI can attach to the cell membrane and interfere with the cellular transport mechanisms. To overcome the PEI’s potential cytotoxicity based on cell membrane attachment, the surface charge of the polyplexes is neutralized. The biotinylated polyplexes were fully coated with streptavidin alone at a PEI to streptavidin ratio of 1 : 1. It was found that nucleic polyplexes lost their non-specific binding to the cell membrane and hence also the non-specific uptake when the surface moiety (in this case, streptavidin) has a near neutral charge at physiological pH (FIG. 7), thereby eliminating their potentially cytotoxic interactions with cells. To validate that the size of these fully coated polyplexes is still within the size range of the CD22 receptor, which is < 150 nm, the size of these polyplexes is measured using Dynamic Light Scattering (DLS) instrument and transmission electron microscope (TEM).

[0130] PEI can also be cytotoxic inside the cell due to its non-biodegradability and its ability to crosslink many negatively charged macromolecules such as polynucleotides and proteins and render them non-functional. To address the PEI’s potential cytotoxicity inside the cell, PEI can be replaced with (or substantially replaced with) biodegradable and less-toxic polymers such as poly (P-amino ester), protamine sulfate (salmine), or chitosan to complex the polynucleotides. Protamine is an arginine-rich protein known to enhance transfection by promoting nuclear transport of transgenes using the 4 nuclear localization sequences (NLS) in its protein sequence. Further, different combinations of low molecular weight (LMW) PEIs such as 0.8, 2, or 5 kDa PEI, and high molecular weight (HMW) PEIs such as 22 kDa linear, 25 kDa branched, or 70 kDa branched PEIs are tested to generate effective transfection reagents. LMW PEIs are known to beAttorney Docket No: LLU 24-009 (105781.0238.0) less toxic than HMW PEIs, while LMW PEIs often give larger nucleic polyplexes than ETMW PEIs.Generating polyplexes with a diametrical size of 150 nm or less

[0131] Nucleic polyplexes of PEI and GFP-expressing plasmids (pGFPs) prepared in pure water are between 75 and 150 nm in size. However, their resuspension in serum-containing media would enlarge the nucleic polyplexes and is therefore expected to enlarge in blood upon intravenous injections. Smaller plasmids offer better transfection / transduction efficiencies than larger ones. Most nucleic polyplexes have a low poly dispersity index (PDI). Based on these results, each nucleic polyplex may have one plasmid, with smaller plasmids giving smaller complexes. For these reasons, different lengths of plasmids (2 to 10 kb) and various cationic polymers including LMW PEIs at different weight ratios or N / P ratios that can together offer the smallest polyplex size, tighter complexes, and resist changes to salt conditions and serum proteins when resuspended in serum-containing culture media are studied using agarose gel retardation assay, SyBr exclusion assay, and DLS measurements.Development and evaluation of B cell transfection reagent

[0132] Based on the high availability of CD22 on the surface of B-ALL cell lines and patient samples (FIG. 4), test different PNP particles (size < 150 nm) are tested with an anti-hCD22 antibody versus an isotype control IgG. The level of the surface coating of these PNPs with CD22 antibody dictates the rate of cargo internalization and thereby the efficacy of transfection. One biotin per protein is attached to an antibody. In addition, to prevent cross-linking of PNPs, the biotinylation levels of the polymer is developed. Different biotinylation ratios on the cationic polymer, and various polymer to streptavidin to antibody ratios to coat the PNP surface are tested. Biotin is quantified using the Pierce Biotin Quantification Kit. Using DLS, the formulation isAttorney Docket No: LLU 24-009 (105781.0238.0) developed in the context of size (Dh), net surface charge, and polydispersity index (PDI) The morphology and size of the functionalized PNPs is validated by TEM imaging.Example 2: Delivery of polynucleotides specifically to CD22+cells in vitro and in vivo

[0133] To generate PNPs, 25 kDa branched PEI was used, which is a nucleic acid carrier used effectively in many gene delivery systems. In this design, PEI was biotinylated using NHS-PEG4- Biotin (NPB). The PEG moieties from NPB have a multipurpose role in that they not only provide water solubility and act as a crosslink between polyplexes and antibodies, preventing steric hindrance, but also provide excellent stealth protection from immune cells when the formulation is intravenously injected into mice. Nucleic polyplexes (FIG. 3) were generated using biotinylated PEI and pGFPs and surface-coated them with conjugates of streptavidin and biotinylated (~1 biotin) antibodies. Effective polyplexes prepared in pure water had a diametrical size within 120- 150 nm (FIGs. 6A & 6B, blue bars). In addition, a reduction in the net charge of the nucleic polyplexes was detected (FIG. 6C) supporting that functionalization is successful and that these can be internalized when binding to the CD22 receptors on cells similar to SIOPs (150 nm, FIGs.5A & 5B)

[0134] For the preparation of monodisperse PNPs and to avoid crosslinks, antibodies are used with 1 (ideal) biotin per protein molecule. For this, the chemical biotinylation of antibodies was developed using NPB and found that the 1 :5 molar ratio gives around 1 biotin per IgG (FIG. 8). Enzymatically biotinylated CD22 antibodies with only one biotin near the C-terminus may also be used. Next, it was confirmed that the biotinylated CD22 antibodies can deliver AF488-labeled streptavidin as a sole cargo to CD22+B ALL cell lines (FIGs. 9A & 9B). PNPs were generated using biotinylated PEI and GFP-containing plasmids (pGFPs) and surface-coated them withAttorney Docket No: LLU 24-009 (105781.0238.0) conjugates of streptavidin and CD22 or IgG antibodies. The PNPs prepared in pure water have a diametrical size of approximately 125 nm (FIG. 6B).In vitro testing of the nucleic acid delivery system

[0135] An empty expression vector such as pUC19 is chemically labeled with Fluorescein (green emission fluorophore) to confirm whether the transfection reagent can deliver the pDNA specifically to ALL cells via the CD22 receptor. Next, the amount of pDNA internalized via anti- CD22 versus IgG antibodies is compared using trypan blue to quench the extracellular green fluorescence during flow cytometric measurements. The concentration of the transfection reagent is developed based on the amount of polymer that is internalized and its cellular effects, in terms of the amount of pDNA per every million ALL cells. After validating the delivery of pDNA, ALL cell lines and ALL patient samples are transfected using pGFP. The transfection efficiencies (GFP+ cell percentage) are measured while checking the cell viability using DAPI via flow cytometry.

[0136] DYRK1 A and BCL2 shRNAs are validated in KMT2A-r ALL cell line. U6 promoter- driven DYRK1A and BCL2 shRNA cassettes are generated using pGFP for CD22-based transfection.In vivo testing of the nucleic acid delivery system

[0137] The tolerability and toxicity of the CD22 PNPs provided herein is tested by intravenously injecting 3 mice / treatment condition with DYRKlA-shRNA or BCL2-shRNA formulation at 0, IX, 2X, and 4X concentrations for a period of 4 weeks (24 mice total). During the 4-week treatment period, changes in mouse weight and activity are checked daily. After the 4- week treatment is completed, the mice are sacrificed and the CD19 / CD22 positive B cells are isolated from the mouse spleens via cell sorting. A reduction in the number of CD19 / CD22 positiveAttorney Docket No: LLU 24-009 (105781.0238.0) cells is thus validated. Reduction of DYRK1 A and BCL2 levels by the PNPs is also validated via western blotting.

[0138] Once the safest and most potent dosage to use is determined, the benefits of the DYRK1A and BCL2 PNPs are tested in 6 distinct in vivo studies, and are compared directly to GNF2133 (DYRK1 A inhibitor) and venetoclax (BCL2 inhibitor). One (1) million cells are injected via tail vein into 56 NSG mice / tested ALL sample. Animals are randomized to the following groups with 7 mice each: 1) vehicle, 2) GNF2133, 3) venetoclax, 4) GNF2133 + venetoclax, 5) control-shRNA, 6) DYRKlA-shRNA, 7) BCL2- shRNA, and 8) DYRKlA-shrNA + BCL2- shRNA. One KMT2A-r ALL cell line (SEM) and one Ph-like ALL cell line (MUTZ5) labeled with luciferase are used to monitor leukemia development during the treatment. Furthermore, two KMT2A-r ALL PDX and two Ph-like PDX cases are used. For the PDX testing, the leukemia development is monitored via weekly blood collection and flow cytometric analysis of CD19 / CD22. 72h after ALL transplantation, mice are treated daily for 4 weeks and sacrificed at the planned study endpoint, which is defined by the presence of > 80% of CD19 / CD22+ALL cells in the blood. Bone marrow and spleen cells are harvested and cell counting, flow cytometric quantification of human CD19 / CD22+ALL cells, and western blotting are performed to test for DYRK1 A, BIM, MYC, and pERK expression levels. Furthermore, Kaplan Meier survival analysis is performed to visualize survival differences between the treatment groups. Comparing the different inhibitors to the PNPs enables assessment of the efficacy of the treatment strategy.Example 3: Polyplex formulation development and characterization

[0139] ATTO488:crRNA polyplexes were prepared using biotinylated PEI (25 kDa) at specified weight ratios. ATTO488-labeled tracrRNA enabled fluorescent tracking of delivery efficiency. Polyplex stability was evaluated in various media conditions including water, RPMIAttorney Docket No: LLU 24-009 (105781.0238.0) complete medium, and blood-mimicking suspension media. Dynamic light scattering (DLS) and zeta potential measurements were performed immediately after preparation and at specified time points. Polyplexes were prepared in different buffer systems (water, acetic acid, sodium acetate buffer) to enhance particle characteristics. The effect of preparation conditions on size, charge, and stability was systematically evaluated.

[0140] As shown in FIGs. 10A-11B, highly efficient delivery of tracrRNA-ATTO488:crRNA polyplexes into SEM cells after 24 or 48 hours was demonstrated. Flow cytometry analysis revealed approximately 11.5% and 100% internalization rate of the total associated polyplexes after 24 and 48 hours, respectively, indicating complete cellular uptake of surface-bound particles within 48 hours. The green window analysis confirmed that virtually all polyplexes that associated with cells were successfully internalized, demonstrating the efficiency of the CD22-mediated endocytosis mechanism.

[0141] As shown in FIG. 12A, the size characteristics of IgG-functionalized polyplexes in blood-mimicking suspension media demonstrated that guide RNA polyplexes prepared using biotinylated PEI (25 kDa, w / w 0.5) conjugated to streptavidin and PBAE 447 (w / w 2.0) maintained appropriate size distributions suitable for cellular interaction even in complex biological media. As shown in FIG. 12B, the polyplexes maintained neutral surface charge in water.

[0142] As shown in FIGs. 13A-13B, comparison of polyplex characteristics in water versus RPMI complete media revealed that siRNA polyplexes showed distinct size profiles depending on the suspension medium, with RPMI complete media generally producing more heterogeneous particles compared to water preparations. This finding highlights the importance of media selection for effective polyplex performance.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0143] The relationship between plasmid length and resulting particle size for IgG- functionalized polyplexes shown in FIGs. 14A-14B provides guidance for developing formulations based on the specific nucleic acid cargo.

[0144] As shown in FIGs. 15A-15B and 16, preparation buffer can influence polyplex size and / or charge characteristics. siRNA polyplexes or DNA polyplexes prepared in water, acetic acid, or sodium acetate buffer showed distinct size and charge profiles.

[0145] Blood-mimicking suspension media were prepared to simulate physiological conditions including appropriate salt concentrations, pH, and protein content. Polyplexes were evaluated immediately after preparation and following incubation in these media. IgG- functionalized and non-functionalized polyplexes containing guide RNA (tracrRNA and crRNA duplex) or plasmid DNA (pUC19) were prepared and characterized. Stability was assessed through size distribution analysis and aggregation monitoring. As shown in FIGs. 17A-17B, the size distribution was similar between IgG-functionalized and non-functionalized polyplexes in water, while IgG-functionalized polyplexes had wider size distribution toward smaller sizes in complete RPMI as compared to in water. This tendency was shown in both guide RNA and DNA polyplexes.

[0146] In sum, the formulation studies reveal parameters that influence polyplex performance. The near-complete internalization efficiency demonstrates the potential of the CD22-targeted system for therapeutic applications. Buffer selection is a variable for polyplex formulations. Polyplex characteristics in media resembling blood support the potential for in vivo applications.Attorney Docket No: LLU 24-009 (105781.0238.0)Example 4: Polyplex formulation strategies using polymer components

[0147] Advanced formulations combining biotinylated PEI, streptavidin, PBAE 447, and / or protamine sulfate were developed to enhance nucleic acid condensation and delivery efficiency. Weight ratios were systematically varied to identify effective compositions.

[0148] Gel electrophoresis was used to assess nucleic acid condensation efficiency. SyBr Green staining and signal quantitation provided quantitative measures of condensation effectiveness across different formulation conditions.

[0149] Size distribution, zeta potential, and poly dispersity were measured for all formulations. Mean fluorescence intensity (MFI) of fluorescent DNA stains provided additional insights into polyplex compactness.

[0150] As shown in FIGs. 18A-18D and FIGs. 19A-19C, comprehensive characterization of siRNA polyplexes prepared in acetic acid with variable PBAE 447 ratios demonstrated that PBAE content affected the condensation characteristics (FIGs. 18A and 19A), size distribution (FIGs.18C and 19B), and zeta potential (FIGs. 18B, 18D, and 19C).

[0151] As shown in FIGs. 20A-20C, the incorporation of protamine sulfate addition at w / w ratios of 1.0 or 2.5 produced affected size characteristics and zeta potential measurements of IgG- coated siRNA polyplexes.

[0152] As shown in FIGs. 21A-21B, enhanced siRNA condensation with increasing protamine sulfate ratios was shown through gel electrophoresis (FIG. 21A) and MFI of fluorescent DNA stains (FIG. 21B).Attorney Docket No: LLU 24-009 (105781.0238.0)

[0153] As shown in FIGs. 22A-22B, use of PBAE 447 (w / w 20-50) and PEI25 in polypi exes resulted in different condensation characteristics.

[0154] As shown in FIGs. 23A-23D, PBAE447 ratios affected size, volume, and charge characteristics of DNA (pUC19) polyplexes.

[0155] In sum, adjusting the formulation (such as PBAE447 ratio, inclusion of prolamine sulfite) enables fine-tuning of polyplex characteristics through systematic evaluation of individual components. The polyplex characteristics provided herein ensure effective formulation selection based on multiple performance criteria.Example 5: CD22-targeted delivery in lymphoma cell lines

[0156] CD22 surface expression was analyzed across multiple B-ALL cell lines representing different molecular subtypes, including KM'12A -rearranged (KMT2A-R) and Philadelphia chromosome-like (Ph-like) B-ALL, as well as a Burkitt’s lymphoma cell line. Flow cytometry analysis used standardized antibody staining protocols with appropriate controls.

[0157] TracrRNA-ATTO488 polyplexes were prepared using developed formulations and functionalized with varying ratios of biotinylated anti-CD22 antibody versus control antibodies (IgG or BSA). Delivery efficiency was quantified through flow cytometry analysis of cellular association and internalization. Multiple antibody ratios were tested (100:0, 75:25, 50:50, 25:75, 0: 100 for anti-CD22:control) to establish the relationship between targeting specificity and delivery efficiency.

[0158] As shown in FIG. 24, CD22 surface expression was demonstrated across multiple B- ALL subtypes (KMT2A-rearranged B-ALL cell lines, Ph-like B-ALL cell lines) and the Burkitt's lymphoma cell line.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0159] As shown in FIGs. 25A-25C, comprehensive validation of CD22-mediated delivery using tracrRNA-ATTO488 polyplexes in SEM-Cas9 cells was achieved. As shown in FIG. 25A, tracrRNA-positive cell ratios increased and cell viability decreased with increasing anti-CD22 antibody ratios. As shown in FIGs 25B and 25C, association and internalization increased with higher anti-CD22 antibody ratios producing enhanced cellular uptake, confirming the specificity of CD22-mediated uptake.

[0160] In sum, the data demonstrate the high affinity and specificity of the CD22 targeting mechanism. CD22 can be a broadly applicable target across CD22 positive cells, including B-ALL subtypes (such as KMT2A-R and Ph-like), and Burkitt’s lymphoma. The dose-dependent delivery enhancement with increasing anti-CD22 antibody content provides strong evidence for receptor- mediated uptake mechanisms.Example 6: Alternative conjugation strategies for antibody functionalization

[0161] An alternative conjugation strategy using direct thiol-pyridyl thiol chemistry was developed to bypass the biotin-streptavidin system. Thiolated PEI was prepared using sulfo-LC- SPDP, and antibodies were modified with pyridyl thiol groups for direct conjugation. TCEP (tris(2-carboxyethyl)phosphine) and PABA (4-aminobenzoic acid) were used to control the thiolpyridyl thiol reaction. Various PELantibody ratios were tested to evaluate conjugation efficiency while maintaining polyplex functionality. The direct conjugation approach was applied to various nucleic acid cargoes including single guide RNA (sgRNA), mRNA, and combination formulations (mRNA plus sgRNA).

[0162] As shown in FIGs. 26A-26B, successful conjugation of IgG to thiolated PEI for siRNA delivery applications was demonstrated. FIGs. 26A and 26B show charge and chargeAttorney Docket No: LLU 24-009 (105781.0238.0) characteristics of siRNA polyplexes prepared using thiolated (-SH) PEI conjugated to IgG via pyridyl thiol (PT), with or without TCEP and PABA to control the reaction between -SEI and PT.

[0163] FIGs. 27A-27C, depict size and charge characteristics of IgG-functionalized polyplexes prepared in water and resuspended in water or RPMI complete media. Polyplexes of single guide RNA (sgRNA) were prepared using unmodified 25kDa PEI (w / w of 1.0) and biotinylated 25kDa PEI (w / w of 1.0) conjugated Streptavidin and to biotinylated IgG (at PEI: IgG = 1 :0.1).

[0164] FIGs. 28A-28C depict size and charge characteristics of IgG-functionalized polyplexes prepared in water. Polyplexes were made using mRNA and 1 / 10 parts of P25-IgG or P25-BSA and 9 / 10 parts of unmodified P25. P25-IgG or P25-BSA were achieved by using thiolated (-SH) 25kDa PEI (thiol generated using sulfo-LC-SPDP) conjugated to IgG or BSA (at PEITgG or PEI:BSA = 1:0.1) via pyridyl thiol (PT) generated using sulfo-LC-SPDP. P25 is 25kDa branched PEI. 2-ME (b-ME) was used to check the aggregation of polyplexes during DLS. As shown in FIGs. 28A and 28B, the formulation affected size distribution of non-reduced and b-ME treated polyplexes. As shown in FIG. 28C non-reduced polyplexes maintained near neutral surface charge, while reduction revealed positive zeta potential in some formulations.

[0165] As shown in FIGs. 29A-29F, detailed analysis of mRNA polyplexes using direct conjugation approaches provided size distributions for thiolated PEI alone (FIG. 29A), IgG- conjugated (FIG. 29B), and BSA-conjugated (FIG. 29C) systems revealing the effects of protein conjugation on particle characteristics. FIG. 29D depicts zeta potential. FIGs. 29E-29F depict size and charge characteristics of non-reduced (FIG. 29E) or reduced (FIG. 29F) polyplexes.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0166] As shown in FIGs 30A-30D, successful formulation of complex nucleic acid combinations including sgRNA, mRNA, and mixed sgRNA+mRNA cargoes was demonstrated. FIGs. 30A-30D depict size and charge characteristics of IgG-functionalized polyplexes prepared in water and resuspended in water. Polyplexes of single guide RNA (KMT2A sgRNA) or mRNA (GFP mRNA) or mRNA plus sgRNA (msgRNA) were prepared using biotinylated 25kDa PEI (w / w of 2.0) conjugated Streptavidin and to biotinylated IgG (at PEI: IgG = 1 :0.1). w / w is calculated as the weight of polymer over the weight of nucleic acid.

[0167] As shown in FIG. 31, specific application to CRLF2 sgRNA delivery demonstrated larger particle sizes likely due to sgRNA secondary structure complexity. This finding highlights the importance of cargo-specific evaluation for different therapeutic targets.

[0168] In sum, the direct conjugation approach provides a viable alternative to biotinstreptavidin linking, offering potential advantages in terms of reduced system complexity and cost. The successful application across multiple nucleic acid types (siRNA, sgRNA, mRNA) and combination formulations demonstrates the versatility of this approach. The comprehensive characterization reveals the importance of understanding disulfide bond contributions to polyplex stability and the need for cargo-specific evaluation based on nucleic acid secondary structure considerations.Example 7: Therapeutic efficacy of D YRKIA -targeting siRNA delivery

[0169] DYRKIA plays a role in B-ALL cell survival and proliferation. Anti-DYRKl A siRNA sequences were designed and incorporated into CD22-targeted polyplexes for specific delivery to B-ALL cells. Multiple siRNA doses (0.5 and 1 fmol / cell) were tested to establish effective therapeutic concentrations. Different antibody sources (BioLegend and Sinobiological) wereAttorney Docket No: LLU 24-009 (105781.0238.0) compared to validate targeting specificity. Knockdown efficiency was assessed by quantitative PCR analysis of DYRK1A mRNA levels. Apoptosis induction was monitored by flow cytometry to evaluate therapeutic efficacy. Cells were treated with siRNA polyplexes in serum-free RPMI for 4 hours followed by addition of complete medium to enhance delivery conditions while maintaining cell viability.

[0170] FIGs. 32A and 32B depict effect of anti- / N7? 7 / l targeting siRNA polyplexes in SEM cells. Cells and formulation were suspended in RPMI complete media. As shown in FIG. 32A, at 0.5 fmol / cell, no significant knockdown efficiency was observed compared to controls. However, as shown in FIG. 32B, at 1 fmol / cell, efficient knockdown was achieved, establishing effective therapeutic applications. Both BioLegend (BL) and Sinobiological (SB) antibody sources produced comparable results.

[0171] As shown in FIGs. 33A and 33C, apoptosis analysis revealed no significant difference in cell death between IgG control and anti-CD22 antibody functionalized polyplexes, but increased apoptosis in anti-CD22 antibody functionalized DYRK1A siRNA polyplexes relative to anti-CD22 antibody functionalized control polyplexes. As shown in FIG. 33B, knockdown efficiency analysis by quantitative PCR demonstrated approximately 33% reduction in DYRK1 A expression in cells treated with anti-CD22 antibody functionalized DYRK1A siRNA polyplexes compared to IgG-functionalized controls. This significant reduction in target gene expression validates the therapeutic potential of the CD22-targeted delivery system.

[0172] In sum, the specificity of the knockdown effect, with enhanced efficacy only observed in CD22-targeted formulations, confirms that therapeutic benefits result from CD22-mediated delivery.Attorney Docket No: LLU 24-009 (105781.0238.0)Example 8: Polyplex Evaluation and Polymer Screening

[0173] Multiple PEI molecular weights (2, 10, 25, 70 kDa) and protamine were systematically evaluated for nucleic acid condensation efficiency and polyplex formation. Both siRNA and plasmid DNA cargoes were tested across the polymer range. Two complementary assays were employed: (1) SyBr exclusion assay to measure nucleic acid accessibility and condensation efficiency, and (2) gel retardation assay to directly visualize nucleic acid migration and complexation. IgG functionalization was developed across a wide range of PELIgG ratios (1 :0.01 to 1: 1) to determine the effects of antibody density on polyplex characteristics and nucleic acid condensation.

[0174] As shown in FTG. 34A, gel retardation assay confirmed complete siRNA condensation across all tested IgG ratios from PEI:IgG 1 :0.01 to 1 :1. As shown in FIG. 34B, dynamic light scattering analysis revealed that the siRNA polyplex sizes were smaller with smaller IgG ratios up to a certain point, indicating the possibility of successful transfection of siRNA into host cells.

[0175] As shown in FTG. 35, PEI molecular weight and ratios affected condensation efficiency for both siRNA polyplexes and plasmid DNA polyplexes. For the 4.7 kb plasmid DNA polyplexes and siRNA polyplexes tested, PEI25 (25 kDa) at w / w 0.5 was sufficient for complete condensation, establishing the effective polymer concentration for large nucleic acid cargoes.

[0176] As shown in FIGs. 36A-36F, systematic comparison of different polymers for siRNA condensation was provided. SyBr exclusion assays for 2 kDa PEI (FIG. 36A), 10 kDa PEI (FIG. 36B), 25 kDa PEI (FIG. 36C), 70 kDa PEI (FIG. 36D), and protamine (FIG. 36E), and gel retardation assay (FIG. 36F), indicate efficient condensation and compactness of siRNA polyplexes at low weight ratios.Attorney Docket No: LLU 24-009 (105781.0238.0)

[0177] In sum, antibody functionalization maintains nucleic acid condensation across a wide ratio range provides formulation flexibility for developing targeting specificity. Formulation can be selected balancing effectiveness with practical considerations.

[0178] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0179] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Claims

Attorney Docket No: LLU 24-009 (105781.0238.0)CLAIMSWhat is claimed is:1 . A composition comprising a complex containing a polynucleotide of interest and a carrier, the surface of the complex being coated covalently or non-covalently with a CD22 receptor ligand.

2. The composition of claim 1, wherein the carrier is a polymer, a lipid, or a protein.

3. The composition of claim 2, wherein the carrier is a cationic polymer.

4. The composition of claim 2, wherein the polymer is polyethyleneimine (PEI), poly-1- lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.

5. The composition of claim 2, wherein the protein is a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.

6. The composition of any one of claims 1 to 5, wherein the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation.

7. The composition of claim 6, wherein the complex is biotinylated.

8. The composition of claim 7, wherein the complex comprises N-hydroxysuccinimide (NHS)-PEG-biotin.

9. The composition of any one of claims 6 to 8, wherein the CD22 receptor ligand is biotinylated and is bound to the streptavidin.

10. The composition of any one of claims 1 to 5, wherein the complex is bound to the CD22 receptor ligand via thiol-pyridyl thiol conjugation.Attorney Docket No: LLU 24-009 (105781.0238.0)11 . The composition of claim 10, wherein the polymer is thiolated and the CD22 receptor ligand is modified to comprise pyridyl thiol.

12. The composition of any one of claims 1 to 11, wherein the CD22 receptor ligand is an anti-CD22 antibody.

13. The composition of claim 12, wherein the anti-CD22 antibody is an anti-CD22 singlechain variable fragment (scFv) antibody.

14. The composition of any one of claims 1 to 13, wherein the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).

15. The composition of any one of claims 1 to 14, wherein the polynucleotide of interest comprises an inhibitory nucleotide against DYRK1 A and / or BCL2.

16. The composition of claim 15, wherein the polynucleotide of interest comprises a pDNA comprising an expression cassette for DYRK1 A and / or BCL2 shRNA.

17. The composition of any one of claims 1 to 16, wherein the polynucleotide of interest comprises a pDNA comprising: an expression cassette for overexpression of BIM and / or PUMA; an expression cassette for shRNA against RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or an expression cassette for shRNA against an upstream or downstream pathway gene target of any one of RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6.

18. A method of delivering a polynucleotide of interest to a B cell, the method comprising: contacting the B cell with a composition comprising a complex comprising the polynucleotide of interest and a carrier, the surface of the complex coated covalently or non- covalently with a CD22 receptor ligand.Attorney Docket No: LLU 24-009 (105781.0238.0)19. The method of claim 18, wherein the carrier is a polymer, a lipid, or a protein.

20. The method of claim 18, wherein the carrier is a cationic polymer.

21. The method of claim 19, wherein the polymer is polyethyleneimine (PEI), poly-l-lysine(PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof; and / or wherein the protein is a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.

22. The method of any one of claims 18 to 21, wherein the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation.

23. The method of claim 22, wherein the complex is biotinylated.

24. The method of claim 23, wherein the complex comprises N-hydroxysuccinimide (NHS)- PEG-biotin.

25. The method of any one of claims 22 to 24, wherein the CD22 receptor ligand is biotinylated and is bound to the streptavidin.

26. The method of any one of claims 18 to 21, wherein the complex is bound to the CD22 receptor ligand via thiol-pyridyl thiol conjugation.

27. The method of claim 26, wherein the polymer is thiolated and the CD22 receptor ligand is modified to comprise pyridyl thiol.

28. The method of any one of claims 18 to 27, wherein the CD22 receptor ligand is an anti- CD22 antibody.

29. The method of claim 28, wherein the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.Attorney Docket No: LLU 24-009 (105781.0238.0)30. The method of any one of claims 18 to 28, wherein the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cast 2-based guide RNA (gRNA), and small interfering RNA (siRNA).

31. The method of any one of claims 18 to 30, wherein the polynucleotide of interest comprises an inhibitory nucleotide against DYRK1A and / or BCL2.

32. The method of claim 31, wherein the polynucleotide of interest comprises a pDNA comprising an expression cassette for DYRK1 A and / or BCL2 shRNA.

33. The method of any one of claims 18 to 32, wherein the polynucleotide of interest comprises a pDNA comprising: an expression cassette for overexpression of BIM and / or PUMA; an expression cassette for shRNA against RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; or an expression cassette for shRNA against an upstream or downstream pathway gene target of any one of RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6.

34. A method of treating a B cell-associated disease in a subject, the method comprising: administering to the subject a composition comprising a complex comprising a polynucleotide of interest and a carrier, the surface of the complex coated covalently or non- covalently with a CD22 receptor ligand, thereby treating the B cell-associated disease in the subject.

35. The method of claim 34, wherein the carrier is a polymer, a lipid, or a protein.

36. The method of claim 35, wherein the carrier is a cationic polymer.

37. The method of claim 35, wherein the polymer is polyethyleneimine (PEI), poly-l-lysine(PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS),Attorney Docket No: LLU 24-009 (105781.0238.0) chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof; and / or wherein the protein is a histone, a histone-like protein, a polynucleotide binding protein, a biologically modified form of any thereof, or combination of any thereof.

38. The method of any one of claims 34 to 37, wherein the complex is bound to the CD22 receptor ligand via streptavidin-biotin conjugation.

39. The method of any one of claims 34 to 37, wherein the complex is bound to the CD22 receptor ligand via thiol-pyridyl thiol conjugation.

40. The method of any one of claims 34 to 39, wherein the CD22 receptor ligand is an anti- CD22 antibody.

41. The method of claim 40, wherein the anti-CD22 antibody is an anti-CD22 single-chain variable fragment (scFv) antibody.

42. The method of any one of claims 34 to 41, wherein the polynucleotide of interest comprises one or more of plasmid DNA (pDNA), linear double stranded DNA (dsDNA), linear single stranded DNA (ssDNA), messenger RNA (mRNA), small hairpin RNA (shRNA), micro RNA (miRNA), transfer RNA (tRNA), CRISPR cas9- or cas 12-based guide RNA (gRNA), and small interfering RNA (siRNA).

43. The method of any one of claims 34 to 42, wherein the polynucleotide of interest comprises an inhibitory nucleotide against DYRK1A and / or BCL2.

44. The method of claim 43, wherein the polynucleotide of interest comprises a pDNA comprising an expression cassette for DYRK1 A and / or BCL2 shRNA.

45. The method of any one of claims 34 to 44, wherein the polynucleotide of interest comprises a pDNA comprising: an expression cassette for overexpression of BIM and / or PUMA; an expression cassette for shRNA against RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6; orAttorney Docket No: LLU 24-009 (105781.0238.0) an expression cassette for shRNA against an upstream or downstream pathway gene target of any one of RAS, MYC, BCR, ABL1, KMT2A, CRLF2, AFF4, AFF9, BRAF, and / or BCL6.

46. The method of any one of claims 34 to 45, wherein the B-cell associated disease is acute lymphoblastic leukemia.

47. A method of preparing a CD22-targeted polyplex composition, the method comprising: contacting a polynucleotide with one or more polymers to form a complex; and binding the complex with a CD 22 receptor ligand.

48. The method of claim 47, wherein the one or more polymers are polyethyleneimine (PEI), poly-l-lysine (PLL), polyethylene glycol (PEG), polydimethylaminoethyl methacrylate (PDMAEMA), poly(amidoamine) (PAMAM), poly (P-amino ester) (PBAE), protamine sulfate (salmine, PS), chitosan (CS), cyclodextrin (CD), a chemically or biologically modified form of any thereof, or combination of any thereof.

49. The method of claim 48, wherein the one or more polymers are 25 kDa PEI and PBAE447.

50. The method of claim 47 or 48, comprising binding the complex with the CD22 receptor ligand via streptavidin-biotin conjugation.

51. The method of claim 47 or 48, comprising binding the complex with the CD22 receptor ligand via thiol-pyridyl thiol conjugation.

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