{"id":612220,"date":"2024-06-11T20:00:00","date_gmt":"2024-06-12T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/transient-growth-factor-expression-via-mrna-in-lipid-nanoparticles-promotes-hepatocyte-cell-therapy-in-mice-nature-communications\/"},"modified":"2024-06-12T13:15:21","modified_gmt":"2024-06-12T17:15:21","slug":"transient-growth-factor-expression-via-mrna-in-lipid-nanoparticles-promotes-hepatocyte-cell-therapy-in-mice-nature-communications","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/transient-growth-factor-expression-via-mrna-in-lipid-nanoparticles-promotes-hepatocyte-cell-therapy-in-mice-nature-communications\/","title":{"rendered":"Transient growth factor expression via mRNA in lipid nanoparticles promotes hepatocyte cell therapy in mice – Nature Communications","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
<\/div>\n

Chronic and acute liver injury mouse models<\/h3>\n

All animal studies were approved by the Boston University IACUC and were consistent with local, state, and federal regulations as applicable. Animals were housed under standard conditions with a 12-hour day\/night cycle in a pathogen-free environment with access to food and water ad libitum. All mice used in Fig. 1<\/a> were male and ranged from 7 to 10 weeks old. All mice used for xenotransplantation studies in Figs. 2<\/a>\u20134<\/a> were male and 7\u20138 weeks old at the time of transplantation. For chronic injury, the transgenic NOD.Cg-Prkdcscid<\/sup> Il2rgtm1Wjl<\/sup> Tg(SERPINA1*E342K)#Slcw\/SzJ (NSG-PiZ) colony was used, mimicking human AATD-associated liver disease. NSG-PiZ mice are homozygous for a transgene expressing the human mutated PiZ allele. For acute liver injury, immunodeficient NOD.Cg-Prkdcscid<\/sup> Il2rgtm1Wjl<\/sup>\/SzJ (NSG) mice were used. Following a 14\u2009h fast to induce consistent liver damage among the mice, liver injury was induced with a single intraperitoneal injection of acetaminophen (N-acetyl-para-aminophenol, paracetamol, APAP) at 200\u2009mg\/kg diluted in sterile PBS (Spectrum Chemical, AC100). Mice were maintained on a normal chow diet and water ad libitum after APAP injections. Both mouse strains were obtained from The Jackson Laboratory (JAX stock #028842 and #005557). As soon as NSG or NSG-PiZ mice were to be used for cell transplantation, they were treated with sulfamethoxazole\/trimethoprim (Sulfatrim) in sterile drinking water on alternate weeks as previously described52<\/a><\/sup>. This antibiotic-supplemented water was given at a dose of 0.6720\u2009mg\/mL drinking solution, protected from light. The water was changed, and fresh antibiotic-supplemented water was given every three days on alternate weeks.<\/p>\n

In vivo administration of AAV8-TBG-p21 and AAV8-TBG-Null<\/h3>\n

The AAV8.TBG.PI.p21.WPRE.bGH vector was obtained from the Penn Vector Core. The AAV8.TBG.PI.Null.bGH vector was obtained from Addgene (105536-AAV8). Mice were anesthetized with isoflurane, and the vectors were thawed, diluted in sterile PBS, and injected retro-orbitally at a dose of 5.00\u2009\u00d7\u20091011<\/sup> gc\/mouse using 29\u2009G Exel International Insulin Syringes (0.5\u2009mL).<\/p>\n

PHH source and transplantation<\/h3>\n

All the following protocols were carried out using aseptic technique in ABSL-2 hoods and using sterile supplies. Cryopreserved PHHs (BioIVT, Lot DJW) were stored at 150\u2009\u00b0C, thawed, and added immediately to 50\u2009mL warm INVITROGRO CP Medium (BioIVT Z990003). The tube was inverted three times slowly to resuspend cells. The cell suspension was centrifuged at 50\u2009\u00d7\u2009g<\/i> for 5\u2009min at room temperature. The supernatant was discarded, and PHHs were resuspended in 5\u2009mL of Hank\u2019s Balanced Salt Solution (HBSS, Gibco 14175095), counted using trypan blue method, and centrifuged again. The supernatant was removed, and cells were resuspended and aliquoted to administer 1 million cells per mouse in ~50\u2009\u00b5L HBSS. Cells were kept on ice and immediately transplanted via intrasplenic injection as previously described53<\/a><\/sup>. Mice were anesthetized with a ketamine\/xylazine cocktail via intraperitoneal injection. BuprenorphineSR painkiller (0.5\u20131.0\u2009mg\/kg) was administered subcutaneously to provide 72\u2009h of analgesia. Ophthalmic ointment was applied to the eyes using the sterile cotton-tipped applicator. Fur was shaved with an electric razor around the spleen area on the left side. Disinfection of the surgical site was performed with iodine pads (Dynarex\u2122 1108) followed by alcohol wipes, repeated three times. Mice were kept warm with a heating pad under the tail and transferred to the sterile field. Sterile drapes, gloves, and surgical instruments were used. An incision of 1\u2009cm was made with a small scissor on the mouse\u2019s left paralumbar fossa to expose the spleen out of the body. The spleen was kept moist with PBS. One million PHHs resuspended in 50\u2009\u00b5L of HBSS were injected into the inferior pole of the spleen using a 27\u2009G tuberculin syringe with an attached needle (BD 305620). Surgifoam gelatin sponge (Ethicon) was used to achieve hemostasis by pressuring a small piece on the injection site for 30\u2009s. The exposed spleen was quickly returned to the abdominal cavity. The body wall was closed with an absorbable suture (Med Vet International VR494), and the skin was closed with 2 wound clips (Reflex, 7\u2009mm). Mice were observed post anesthesia until they awoke.<\/p>\n

mRNA production<\/h3>\n

mRNAs were produced using T7 RNA polymerase (Megascript, Ambion) on linearized plasmids encoding codon-optimized firefly luciferase (Luc)54<\/a>,55<\/a><\/sup>, eGFP, HGF, and EGF. mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. One-methylpseudouridine (m1\u03a8)\u22125\u2019-triphosphate (TriLink) instead of UTP was used to generate modified nucleoside-containing mRNA. RNAs were capped using the m7G capping kit with 2\u2019-O-methyltransferase (ScriptCap, CellScript) to obtain cap1. mRNAs were purified by cellulose-based purification as described56<\/a><\/sup>. All mRNAs were analyzed by agarose gel electrophoresis and were stored frozen at \u221220\u2009\u00b0C. The optimized coding sequences of DNA for making nucleoside-modified mRNA are listed in Supplemental Table 1<\/a>.<\/p>\n

LNP formulation of the mRNA<\/h3>\n

FPLC-purified m1\u03a8- containing mRNAs were encapsulated in LNP using a self-assembly process in which an aqueous solution of mRNA at pH 4.0 was rapidly mixed with a solution of lipids dissolved in ethanol57<\/a><\/sup>. LNP used in this study contains an ionizable cationic lipid (pKa in the range of 6.0\u20136.5, proprietary to Acuitas Therapeutics)\/distearoylphosphatidylcholine\/cholesterol\/PEG-lipid57<\/a>,58<\/a><\/sup>. The proprietary lipid and LNP composition are described in US patent US10,221,127 entitled \u201cLipids and lipid nanoparticle formulations for delivery of nucleic acids\u201d (https:\/\/www.lens.org\/lens\/<\/a> patent\/183-348-727-217-109)59<\/a><\/sup>. They had a diameter of ~80\u2009nm as measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) instrument. Acuitas will provide the LNP used in this work to academic investigators who would like to test it.<\/p>\n

In vivo administration of mRNA-LNP<\/h3>\n

mRNA-LNP were thawed and freshly diluted on ice in sterile Dulbecco\u2019s phosphate-buffered saline (DPBS) prior to each experiment. Mice were anesthetized with isoflurane and administered 50\u2009\u03bcL of diluted mRNA-LNP encoding luciferase (10\u2009\u03bcg\/mouse), eGFP (10\u2009\u03bcg\/mouse), or combination HGF (5\u2009\u03bcg\/mouse) + EGF (5\u2009\u03bcg\/mouse) intravenously by retro-orbital injection using 29\u2009G Exel International Insulin Syringes (0.5\u2009mL).<\/p>\n

EdU injection, animal sacrifice, serum collection, liver tissue fixation, cryopreservation, and sectioning<\/h3>\n

2\u2009h prior to sacrifice, mice were injected intraperitoneally with 200\u2009\u03bcL of EdU solution at 5\u2009mg\/mL in sterile DPBS (Sigma Aldrich 90058450MG). Euthanasia was performed at indicated end points by CO2<\/sub> inhalation, after which death was determined by cessation of breathing, lack of pulse, and limb pinching. Then, cervical dislocation was performed. This method of euthanasia is consistent with the recommendations of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Whole blood samples were collected from the inferior vena cava and allowed to clot at room temperature for 15\u2009min. Then, blood samples were centrifuged at 1000\u20132000\u2009\u00d7\u2009g<\/i> for 15\u2009min at room temperature. The serum layer was separated and kept at \u221280\u2009\u00b0C until required for analysis. Liver and spleen tissues were harvested and fixed in 4% paraformaldehyde (Electron Microscopy Sciences 15710) solution overnight at 4\u2009\u00b0C. The next day, the tissues were placed in a 15% sucrose solution for 30\u2009min, and then placed in a 30% sucrose solution for at least 48\u2009h. Fixed liver and spleen tissues were flash frozen into OCT (Fisher 23-730-571) blocks and sectioned using a cryostat (CM1950 Leica) at 5\u2009\u00b5m thickness onto microscope slides (Fisher 12-550-15). Sections were stored at \u221220\u2009\u00b0C until required for immunostaining.<\/p>\n

Immunohistochemistry on PFA-fixed frozen tissue sections<\/h3>\n

The frozen sections were allowed to defrost and dry at room temperature for 10\u2009min. The slides were dipped in PBS for 10\u2009min, then permeabilized using 0.3% triton X in PBS for 10\u2009min. The slides were rinsed three times in PBS, 10\u2009min each, and blocked with 3% normal donkey serum for 30\u2009min. The sections were then incubated overnight at 4\u2009\u00b0C with appropriate primary antibodies or antibodies diluted in PBS with 0.1% BSA at concentrations indicated in Supplemental Table 2<\/a>. For nuclear staining with p21 and Ku80, 0.2% triton X was also included in the overnight primary antibody incubation. For EGF and 2C1 staining, the Mouse on Mouse kit (Vector Labs, BMK-2202) was used to reduce background from mouse tissue. Following primary antibody incubations, the slides were washed three times with PBS, 10\u2009min each. Then slides were incubated with the appropriate fluorescent labeled secondary antibodies for 1\u2009h at room temperature, protected from light. The slides were washed three times in PBS for 10\u2009min each. Finally, the slides were incubated with DAPI (1:3000 in PBS) for 3\u2009min, washed three times in PBS, and mounted using FluorSave reagent (MilliporeSigma, 345789-20\u2009mL) and a coverslip (Epredia, 102450). For EdU detection, staining was performed using Click-iT EdU Imaging Kit (Invitrogen C10337) after the secondary antibody incubation step following the manufacturer\u2019s protocol. The Invitrogen Click-iT Plus TUNEL Assay Kit for In Situ Apoptosis Detection (Invitrogen, C10617) was used to stain apoptotic and necrotic cells.<\/p>\n

PAS-Diastase stain<\/h3>\n

Periodic acid Schiff (PAS) staining stains both glycogen and Z-AAT globules. Diastase digests away glycogen, allowing for visualization of only misfolded Z-AAT protein. First, PFA-fixed frozen tissue sections were brought to room temperature and hydrated with diH2<\/sub>O for 5\u2009min. The diastase\/a-amylase solution was prepared fresh by adding 0.4\u2009g diastase (Sigma Aldrich, A3176) to 80\u2009mL diH2<\/sub>0, microwaving for 30\u2009s, and mixing gently. Tissue sections were placed in the warmed diastase solution for 45\u2009min. Slides were washed in several changes of diH2<\/sub>O, followed by PAS staining using a kit (Sigma Aldrich, 395B). Sections were oxidized with a periodic acid solution for 5\u2009min, rinsed in several changes of diH2<\/sub>O, and incubated in Schiff\u2019s Reagent for 15\u2009min. After rinsing in diH2<\/sub>O again, sections were dehydrated and mounted with permanent mounting media.<\/p>\n

Microscopy, image analysis, and quantification<\/h3>\n

All images were taken using a Nikon microscope (Nikon Eclipse Ni-E) and analyzed using NIS Elements software and Fiji. For all histology quantification, measurements for each mouse were averaged using three 10\u00d7 images per mouse, each from a different liver lobe. For quantifying EdU+<\/sup> or p21+<\/sup>hepatocytes, positive cells were counted manually based on morphology using high power magnification. To quantify the number of PHH clusters\/field, cells\/cluster, and EdU+<\/sup> cells\/cluster, positive events were manually counted per image using high-power magnification. To quantify the percent hALB+<\/sup> or 2C1+<\/sup> area, positive areas were measured using Fiji and were divided by the total tissue area, accounting for empty areas due to large vasculature such as portal and central veins. The percentage of liver repopulation was quantified by tracing the area occupied by hALB+ <\/sup>Ku80+<\/sup> PHHs and dividing by total tissue area, accounting for empty areas due to large vasculature such as portal and central veins.<\/p>\n

Enzyme-linked immunosorbent assays (ELISA)<\/h3>\n

Serum samples were stored at \u221280\u2009\u00b0C, thawed on ice, and used immediately. Repeat freeze-thaw cycles were avoided. The following ELISAs were performed according to the manufacturer\u2019s protocols, specifically following the instructions for serum samples: human albumin (Bethyl Laboratories, E88-129), human HGF (Abcam, ab275901), and human EGF (Abcam, ab217772). The total human AAT ELISA was performed using a quantification kit (GenWay Biotech). This protocol was then adapted to include a Z-AAT antibody and standard as described here60<\/a><\/sup>.<\/p>\n

ALT assay<\/h3>\n

Assays were performed using the Pointe Scientific kit (A7526-450) for testing serum ALT levels following manufacturer\u2019s protocol. Briefly, 10\u2009\u03bcL of serum was mixed with supplied reagent mix at 37\u2009\u00b0C and readings were measured at 340\u2009nm every 1\u2009min for 5\u2009min using Molecular Devices SpectraMax i3x Multi-Mode microplate reader.<\/p>\n

Figure generation<\/h3>\n

Cartoons and schematics were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.<\/p>\n

Statistics and reproducibility<\/h3>\n

All statistical analyses were performed using GraphPad Prism version 10.0.0 for Mac, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com<\/a>. For comparison between the two groups, an unpaired two-sided t<\/i> test was used to calculate statistical significance. For comparing multiple groups, an ordinary one-way unpaired ANOVA was performed. Quantitative data are shown as mean\u2009\u00b1\u2009standard error of the mean (SEM) and are considered statistically significant when p<\/i>\u2009<\u20090.05 (ns P<\/i>\u2009>\u20090.05, *P<\/i>\u2009\u2264\u20090.05, **P<\/i>\u2009\u2264\u20090.01, ***P<\/i>\u2009\u2264\u20090.001, ****P<\/i>\u2009\u2264\u20090.0001).<\/p>\n

Reporting summary<\/h3>\n

Further information on research design is available in the Nature Portfolio Reporting Summary<\/a> linked to this article.<\/p>\n