Original Research Proposal Fall 2018

A pH-Responsive Hyaluronic Acid-based Nanogel for the Delivery of Cas9 RNP Complex with Enhanced Endosomal Escape

Department of Chemistry University of Wisconsin-Madison

Committee – Dr. Sandro Mecozzi (PI), Dr. Helen Blackwell, Dr. Glen Kwon

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Summary: The fight against cancer has employed a variety of techniques including chemotherapy1, radiotherapy2, and immunotherapy3. The discovery of clustered regularly interspaced short palindromic repeats (CRISPR) has significantly enhanced the potential of biotherapeutics to combat cancer.4 CRISPR systems are extremely effective at gene editing due to their simplicity and specificity, and therefore can be used in cancer therapy to silence or replace oncogenes.5 However, delivering CRISPR molecules are difficult as they are rapidly cleaved and cleared in the bloodstream. As such, nanotechnology has been employed to deliver CRISPR therapies.6–9 While CRISPR plasmids and/or mRNA are typically delivered, it is safer and more effective to deliver Cas9 ribonucleoprotein complex (Cas9 RNP).10 Yet, Cas9 RNP is not commonly delivered due to the difficulties in loading, release, and endosomal escape (EE).

Recently, small hydrophobic peptides termed endosomal disruptive peptides (EDPs) have been shown to greatly improve the EE of proteins.11 EDPs function by intercalating into the lysosomal lipid bilayer and destabilizing it. This strategy offers a better alternative to toxic polymers such as polyethylenimine.12 However, EDPs have never been used in conjunction with nanoparticles. While EDPs can be used for EE, they do not help with difficulties in loading and release. To combat these problems, nanogels (NGs) have recently emerged as an effective solution.13,14 NGs are porous particles that have an aqueous gel network resembling a tissue environment, which is optimal for delivering biomolecules. NGs are highly tunable and can be engineered to trap biotherapeutics with high loading and fast release by utilizing fast gelation strategies and labile crosslinkers.15 However, there have been no attempts to deliver Cas9 RNP using solely a NG.

This proposal aims to deliver Cas9 RNP with high encapsulation efficiency, active targeting, fast release, and enhanced EE, by synthesizing and assembling a hyaluronic acid NG that contains EDPs. The EDPs will be linked, and the NG will be formed, via strain-promoted click chemistry. Additionally, the linkers and EDPs will contain internal pH-sensitive acetals that allow the NG to decompose, and the EDPs to release, once the NG has reached the acidic lysosome. The efficacy of the NG system will be tested in vitro and in vivo by silencing PD-L1 expression.16 Specific Aims: 1. Synthesis and characterization of a pH-responsive HACO nanogel harboring Cas9-NLSa. The cyclooctyne-functionalized hyaluronic acid (HACO) polymer17, the azido-pH-sensitive linker, and

the azido-pH-sensitive GWWG EDP peptide11 will be synthesized. The EDP peptide will be attached tothe HACO polymer via strain-promoted click chemistry.

b. NGs with and without EDPs will be formulated via inverse nanoprecipitation.18–21 Composition andsize will be evaluated via a combination of FTIR, NMR, nanoparticle tracking analysis, and zetapotential. Toxicity will be gauged by a zebrafish teratology study.22 Encapsulation efficiency will bemeasured using ATTO-labeled scramble Cas9-NLS (ATTO-scrCas9-NLS).

c. The pH-stimulated dissociation of the NGs and release of Cas9-NLS and the EDPs will be assessed byincubating the NGs in different pH solutions and analyzing their dissociation with LCMS. The role ofthe EDPs will be quantified by transfecting 4T1 and A549 cells with NGs encapsulating ATTO-scrCas9-NLS that contain or do not contain EDPs. The number of particles that escape the lysosome will beanalyzed with confocal microscopy.

2. Cellular and animal efficacy studies:a. In vitro - 4T1 and A549 cells will be treated with NGs harboring Cas9-NLS targeting PD-L1. PD-L1

silencing will be measured using RT-qPCR, Western blot, and a SURVEYOR assay.16,23

b. In vivo – NGs containing Cas9 RNP targeting PD-L1 will be injected into the tail vein of 4T1 tumor-bearing mice. Tumor volume, mouse survival time, and NG biodistribution will be assessed.Additionally, PD-L1 expression will be quantified as described above.

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Introduction: Background and Significance: Cancer is the second leading cause of the death in the United States.24 While the overall mortality rate has decreased, the incidence rate for being diagnosed with an aggressive cancer is 38%. Tumor formation, proliferation, and metastasis are often aided or caused by overexpressed proteins. One example is programmed death-ligand 1 (PD-L1), an oncoprotein that is overexpressed on the cell surface of many tumors and causes chemoresistance, suppresses T-lymphocyte immune response, and aids in metastasis16,25,26. While therapeutics such as siRNAs27, small-molecule inhibitors28, and antibodies29 have emerged as effective tools against oncoproteins, these therapies function by targeting the protein itself or the corresponding mRNA. These effects are typically transient and require multiple doses. Therefore, it is more valuable to silence the oncogene completely. This has been made more feasible by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR), a system that can be used for direct editing of the genome.4,5,30–32

CRISPR systems are composed of tracer RNA (tracrRNA), CRISPR RNA (crRNA), and CRISPR associated protein 9 (Cas9). crRNA guides the Cas9 endonuclease to the corresponding section of DNA, while tracrRNA binds to crRNA to activate the Cas9 ribonucleoprotein complex (Cas9 RNP).30 Once led by the RNAs, Cas9 induces a double-strand break that can be repaired via non-homogenous end joining (NHEJ), in which the broken ends are ligated back together, or via homology directed repair (HDR), in which a DNA sequence is fitted in between the broken strands. NHEJ can cause indels that can lead to silencing of the protein expression due to the formation of a premature stop codon. HDR can be utilized to repair proteins by co-transfecting a DNA strand that adds to or replaces the broken strand.

The power of CRISPR lies in the potency, specificity, and ease of gene editing; however, CRISPR molecules, like most biotherapeutics, are readily cleaved in the bloodstream via enzymes such as proteases and nucleases.33 Therefore, nanotechnology has been implemented to protect and deliver CRISPR molecules. Typically, CRISPR is delivered as a plasmid and/or mRNA; yet, these methods suffer from lower editing efficiency, higher off-target effects, and higher immunogenicity.10 One alternative method is to deliver Cas9 RNP, which has been shown to have an improved efficacy and reduced toxicity.34 Still, Cas9 RNP is not commonly delivered due to the large size of the 160 kDa Cas9 protein. Successful delivery has been demonstrated with nanovehicles such as gold nanoparticles34, metal-organic frameworks8, polyplexes35, cationic lipid polymers36, and DNA nanoclews37. Nevertheless, despite these successes, there are still major problems in loading, release, and endosomal escape (EE).38

EE is considered the rate-limiting step for delivering macomolecules.38 Several strategies have been employed to enhance EE of Cas9 RNP, the most common being the proton-sponge hypothesis (PSH). This method typically utilizes polymers with basic nitrogen groups that induce osmotic bursting of lysosomes due to the influx of protons, chlorides anions, and water.39 Unfortunately, a major drawback is that these polymers, including the commonly-used polyethylenimine, are very toxic.40 Additionally, it has been recently reported that EE via the PSH is greatly dependent on the size and leakiness of the endosome – factors that can be drastically different depending on the cell type.41 Rehman et al. reported that cargofrom only one or two LPEI polyplexes per cell could escape the endosome of HeLa cells through the PSH.Therefore, new strategies are needed to increase the EE of nanocarriers. Recently it has been shown thatsmall peptides containing tryptophan can destabilize endosomes through hydrophobic interactions andaid in the delivery of macromolecules.11,42 This type of endosomal disruption is utilized by viruses, such asinfluenza, which uses the hydrophobic section of the protein hemagglutinin to bury into and destabilizethe cell membrane.43 While these endosomal disruptive peptides (EDPs) have been used in conjunctionwith cell penetrating peptides to deliver macromolecules, they have not been used in nanoparticles.11

The most difficult aspect of loading and releasing biotherapeutics is that the nanovehicle is not always compatible with hydrophilic biomolecules. This is often circumvented by editing the cargo to be more compatible, which can decrease efficacy, or by adding polymers to the nanoparticle, which can increase toxicity. To combat these challenges, nanogels (NGs) have recently emerged as effective

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solutions.13,14 NGs are porous particles that have an aqueous gel network typically composed of a polysaccharide and linker. This architecture resembles a tissue environment, which is optimal for delivering biomolecules. Thus, NGs have been used to deliver proteins44, siRNA20, and plasmids45,46. Despite the diversity in cargo however, there have been no attempts to deliver a CRISPR-based system using solely a NG. Due to the highly tunable nature of NGs, they can be engineered to trap biotherapeutics with high loading by utilizing fast gelation techniques. While most of these strategies require harsh UV light or chemical agents, which can damage cargo, these effects can be minimized by using chemical-free bioorthoganol chemistry and nanoprecipitation methodology.15,47

Hypothesis: In considering the advantages of NGs and EDPs, this research proposal describes a novel system that combines the two technologies to deliver Cas9 RNP with enhanced EE. The NGs will be composed of hyaluronic acid (HA), a commonly-used disaccharide that binds to CD-44, a cell receptor overexpressed on a variety of cancers.18,48–50 This will enable the NGs to actively target tumors. HA will be functionalized with a cyclooctyne moiety, while the EDPs and NG linkers will contain azido groups. Strain-promoted click chemistry will then be used to attach the EDPs to HA and to form the NGs. This will preclude the use of toxic reagents to produce the gel. Additionally, the linkers and EDPs will contain internal acetal groups, allowing the NGs to dissociate, and EDPs and Cas9 RNP to be freed upon reaching the acidic environment of the lysosome. The efficacy of the NG system will be tested in vitro and in vivo by targeting PD-L1 expression.

Methodology: 1a. Synthesis of PEGylated linkers, EDPs, and cyclooctyne-substituted HA (HACO) polymer

The linker is designed such that the length will form NGs with pore sizes that allow Cas9 RNP to fit into the NG upon gelation, but unable to passively diffuse out until the NG reaches the lysosome. Since the linker will be composed of two PEG units, PEG5,000 is hypothesized to be a good candidate. This is because the estimated linker length will be ~64 nm, which should form enough space to fit the Cas9 RNP,

Scheme 1

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which has a hydrodynamic radii of ~7.5 nm.10 To form the pH-sensitive linker (Scheme 1), PEG5,000 is monoprotected using one equivalent of benzyl bromide and sodium hydride to form the benzyl-protected PEG (1). The non-protected alcohol is converted to the aldol (2) by reacting it with two equivalents of p-anisaldehyde, using p-toluenesulfonic acid (p-TSA). The benzyl groups are removed using palladium-catalyzed hydrogenation to produce the free alcohol (3). A Williamson ether synthesis is performed using sodium hydride and epichlorohydrin to from the epoxide-functionalized PEG (4). Dilute sodium hydroxide converts the epoxides to diols (5), which are mesylated (6) using mesyl chloride in triethylamine. Finally, sodium azide is used to yield the pH-sensitive azido-linker (7).

The pH-sensitive EDP is synthesized starting with a PEG composed of five units (Scheme 2). This provides a sufficient distance between the EDP and the pH-sensitive moiety, as the latter could impact endosome disruption.11 Triisopropyl silyl chloride (TIPS) and sodium hydride are used to form the monoprotected PEG (8).51 The non-protected end is mesylated (9) using mesyl chloride and then attached to p-anisaldehyde via a Williamson ether synthesis to form the PEGylated benzaldehyde (10). In a separate step, 3-chloro-1,2-propanediol is reacted with sodium azide to form 3-azido-1,2-propanediol (11).52 11 undergoes aldol formation with 10 using p-toluenesulfonic acid to produce the PEGylated azido-acetal (12). The TIPS protecting group is removed with tetrabutylammonium fluoride (TBAF) to form the free alcohol (13), which is then reacted with succinic anhydride and pyridine. Next, the resulting carboxylic acid compound (14) is converted to an N-hydroxysuccinimide (NHS)-ester (15) using N,N'-diisopropylcarbodiimide (DIC) to activate the carboxylic acid. Finally, the NHS-ester is reacted with the N-terminus of the purchased GWWG peptide, resulting in the pH-sensitive azido-EDP (16).53

Scheme 2

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The synthesis of the HACO polymer follows literature conditions (Scheme 3).17,54 First, cycloheptene undergoes a cycloproponation reaction with bromoform, potassium tert-butoxide, and tert-butanol, to form 8,8-dibromobicyclo[5.0.1]octane (17). This is followed by a ring-opening elimination reaction using N-(2-hydroxyethyl)trifluoroacetamide and silver trifluoromethylsulfonate to form (Z)-N-(2-(2-bromocyclooct-2-enyloxy)ethyl)-2,2,2-trifluoroacetamide (18). A subsequent elimination reaction is performed using 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) to afford 2-(cyclooct-2-ynyloxy)-2,2,2-trifluoroacetamide (19). The trifluoroacetamide protecting group is removed using a solution of potassium carbonate in methanol to generate 2-(cyclooct-2-ynyloxy)ethanamine (20). Finally, 20 is dissolved in an acidic 2-(N-morpholino)-ethanesulfonic acid (MES) buffer and then coupled to 60 kDa HA using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), which activates the carboxylic acid of HA. The molar ratio of HA to 20 will be 3:1, which has been shown to lead to a substitution rate of 25%.17 The HACO polymer (21) will be extensively dialyzed with sodium chloride and distilled water, followed by lyophilization to obtain a solid.

HA of 60 kDa was chosen because strong binding to CD-44 requires HA of at least 30 kDa, but higher molecular weight HA may lead to larger NGs.50 The degree of cyclooctyne substitution will be evaluated via 1H-NMR by comparing the integration of the native HA peak at 4.45 ppm with the cyclooctyne peak at 4.28 ppm (Figure 1).17 The HACO will be coupled to the EDPs through strain-promoted click chemistry, simply by mixing 21 in PBS with 16 at a molar ratio of 10:1 HACO:EDP to form the EDP-conjugated HACO (HACO-EDP) (22) (Scheme 4). The success of the click reaction will be evaluated by FT-IR by examining the absence of the azide peak at 2100 cm-1

. Coupling efficiency will be calculated with 13C-NMR by comparing the integration ratio of the alkynepeak at 92 pm to the alkene peak at 165 ppm (Figure 2). 22 will be dialyzed and lyophilized as described above.

OHO

NH

HO

OOHO

OHO

O

O

HN

16

21

O

OHO

NH

HO

OOHO

OHO

O

O

HN

O

NN

N O

O

OO O

HN

GWWG

O

22

5

Scheme 3

Scheme 4

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1b. Nanogel formation and characterization: Cas9 conjugated to a nuclear localization sequence (Cas9-NLS), tracrRNA, and crRNA targeting the

PD-L1 gene will be purchased. The NLS peptide will allow the Cas9 RNP to enter the nucleus. The RNAs and Cas9-NLS protein will be mixed in a 1:1:1 w/w/w ratio in 1X tris-EDTA buffer for 10 min at 37° C to form the Cas9-NLS RNP (PD-Cas9-NLS). A control Cas9 RNP will be purchased that contains a scramble crRNA sequence and tracrRNA conjugated to the fluorescent dye ATTO 550 (ATTO-scrCas9-NLS). The latter will allow fluorescence imaging. The control Cas9 RNP will be assembled in the same manner.

The NGs will be formulated via inverse nanoprecipitation. In this method, aqueous solutions at 4° C of 21 or 22, Cas9 RNP, and 7 are quickly mixed and added to a solution of acetone that is stirring rapidly. The cold temperature prevents premature click reactions in the aqueous solution.21 Two NGs will be formulated that contain the EDP (HACO-EDP-NG) or do not contain the EDP (HACO-NG). The latter will be used as a control to test the impact of the EDPs on EE. Inverse nanoprecipitation forms NGs because HA is insoluble in acetone, so when an aqueous solution of HA is added, water diffuses into the organic solvent leading to supersaturated HA solutions.55 Cas9 RNP also is insoluble in acetone so it remains trapped in the polysaccharide. Since the solution is stirring, islands of supersaturated HA appear, which forms NGs upon interaction with the linkers through strain-promoted click chemistry. This method has been employed ubiquitously to formulate a variety of NGs, including click-based NGs.20,21,45,47,56 After the NGs have precipitated, acetone will be removed in vacuo and the aqueous solution of NGs will be centrifuged, washed, and then lyophilized to obtain a powder. NG size and concentration will be analyzed using nanoparticle tracking analysis.57 Zeta potential will be measured via a Zetasizer instrument.

The toxicity of the HACO-EDP-NG and HACO-NG will be evaluated via an a zebrafish teratology study using NG concentrations of 1 μM, 100 μM, 500 μM, and 1 mM.22,58 Embryos will be evaluated for developmental defects over a 72 h period. Encapsulation efficiency of both NGs will be measured by repeating the NG formulation procedure using ATTO-scrCas9-NLS. After the NGs are centrifuged, washed and removed, the fluorescence of the supernatant will be compared to the fluorescence of the NG. For the latter part, a sample of the solid NGs will be mixed with a hyaluronidase solution, an enzyme that cleaves HA. This will decompose the NGs, freeing the ATTO-scrCas9-NLS. Fluorescence will be measured using an excitation of 554 nm and an emission of 576 nm, and a standard curve will be developed to allow quantification of encapsulation efficiency. If a high encapsulation efficiency (>95%) is not observed, there

Figure 2. The degree of EDP coupling to HACO will be evaluated using the equation 𝑐𝑜𝑢𝑝𝑙𝑖𝑛𝑔 % = ∗

100%, where 𝑐 and 𝑑 correspond to integration values of the labeled carbons.

Figure 1. The degree of cyclooctyne coupling to HA will be evaluated using the equation

𝑐𝑜𝑢𝑝𝑙𝑖𝑛𝑔 % = ∗ 100%, where 𝑎 ,

𝑎 , and 𝑏 correspond to integration values of the labeled hydrogens.

OHO

NH

OH

OOHO

OHO

O

OHN

b - 4.28 ppm

a1

a2

a1 & a2 - 4.45 ppm

O

21

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are two likely causes: either the pore size is too small and the Cas9 RNP cannot physically fit, or the pore size is too large and the Cas9 RNP will diffuse out of the NG. If the pore size is too small, several strategies can be employed, such as increasing the linker length, decreasing the ratio of linker added to the HACO, appending less cyclooctyne moieties to HA, and using a larger HA molecular weight. If the pore size is too large, the opposite strategies will be utilized. Either case also could be solved by fine-tuning the nanoprecipitation methodology, such as by using a different solvent, adjusting the ratios of the components, and using sonication instead of mixing. 1c. Evaluating Cas9 RNP release and endosomal escape

The pH-responsive dissociation of the linkers and EDPs will be evaluated for HACO-EDP-NGs and HACO-NGs via incubating the NGs in solutions of varying acidity. The results will be quantified using reverse-phase LCMS. Separate peaks should be observed for the dissociated EDPs, HA/linker products, and the Cas9 RNP. Additionally, NG dissociation and EE of the NGs will be evaluated using 4T1 mouse breast cancer and A549 human lung carcinoma cell lines. In these studies, the HACO-EDP-NGs and HACO-NGs will be formulated with ATTO-scrCas-NLS and imaged using confocal microscopy. LysoTracker Green and DAPI dyes will be used to verify NG entry into the lysosome and nucleus, respectively. It is expected that the HACO-EDP-NGs will show greater transfection efficiency and entry into the nucleus than the HACO-NGs due to the presence of the EDPs. Depending on the results however, the sensitivity of the acetal groups may need to be adjusted. For example, if the acetal groups are too sensitive to acidic conditions, the NG may decompose in the acidic extracellular matrix of the tumor site, releasing the cargo prematurely. In this case, the benzene ring of the acetal moiety would be functionalized with electron-withdrawing nitro groups, which reduces the sensitivity of the acetal groups to acidic pH.59 Conversely, the acetal groups may decompose incompletely or too slowly to release the cargo, which could be remedied by adding electron donating methoxy groups to the benzene ring of the acetal group, increasing the sensitivity to acidic pH.59 If the GWWG EDP causes low endosomal disruption, other EDPs can be tried, such as GFWFG or GWGGWG.11 2a. In vitro experiments to assess Cas9 RNP efficacy

To test whether NGs harboring PD-Cas9-NLS silence PD-L1, HACO-EDP-NGs containing PD-Cas9-NLS will be incubated in 4T1 and A549 cells at Cas9 RNP concentrations of 10 nM, 50 nM, 100 nM, 500 nM, and 1 μM. PD-L1 silencing will be quantified via RT-qPCR, Western blot and a SURVEYOR assay after 48 h of treatment, using literature conditions.16,32,60 The SURVEYOR assay will examine the degree indels produced via NHEJ on the PD-L1 gene.23 The same cell culture will be analyzed again after 7 days to verify that the PD-L1 gene has been permanently silenced. In each experiment, NGs harboring PD-L1-targeting Cas9 will be compared to NGs containing ATTO-scrCas9. The scramble crRNA sequence prevents Cas9 from inducing double-strand breaks, and therefore will serve as a reliable control. 2b. In vivo experiments to assess Cas9 RNP efficacy

Fifteen BALB/c mice will be injected subcutaneously in the abdomen with a suspension of 4T1 cells. Once tumors have grown to a volume of 50 mm3, the 15 mice will be split into 3 equal groups designated as treatment, scramble, and control. The mice in the treatment group will be injected with HACO-EDP-NGs harboring PD-Cas9-NLS at a concentration of 1 mg of Cas9 RNP per 1 kg of mouse body weight. The mice in the scramble group will be injected with HACO-EDP-NGs harboring ATTO-scrCas9-NLS at the same concentrations. For the control group, the mice will be injected with PBS. All injections will be through the tail vein. Tumor volume will be measured on days 0, 1, 3, 5, 7, and every 7 days until 42 days have been reached. The mice in each group will be sacrificed, and the tumors will be excised and digested to analyze PD-L1 expression as described above. Additionally, tumor metastases will be checked

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for each group by looking for metastatic lesions on bones and lungs.61 Biodistribution of the NGs to the major organs will be evaluated for the scramble group using fluorescence imaging.

Conclusion: The drug delivery of Cas9 RNP has many barriers, with the most difficult being loading, release,

and EE. This proposal addresses these issues by encapsulating Cas9 RNP in a NG composed of an active targeting HA polymer decorated with pH-sensitive EDPs and crosslinked with pH-sensitive linkers. By utilizing this strategy, Cas9 RNP will be released only when the NG reaches the acidic lysosome of a tumor cell. The addition of EDPs will greatly enhance EE, enabling higher gene editing potential. Overall, this nanoparticle overcomes the most challenging barriers of drug delivery by combining proven technologies in a novel fashion.

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