Enhanced Gene Delivery and Expression using Intraosseous Injection of Chitosan Nanoparticles Encapsulated Adenine Base Editor Plasmids

We propose a gene therapy method that directly delivers Adenine based Editor Chitosan to the bone marrow through Intraosseous injection. The transfection efficiency of exogenous plasmids into experimental animals is relatively low and the introduction of plasmids for long-term gene expression may be affected by the immune system. We use the to chitosan to encapsulate Adenine based editor plasmids and directly delivered the complex to the bone marrow of mice through intraosseous injection, making it suitable for diseases caused by abnormal osteoclast function.

Our protocol offers high transfection efficiency, small biological damage, and a high gene expression efficiency. Our novel strategy is not only suitable for diseases caused by abnormal osteoclast function, but also has significant potential for advancing the field of gene therapy. To begin place a euthanized mouse on a work table.

Using surgical scissors and tweezers, remove the tibia and strip away all surrounding muscle tissue. Soak the cleaned tibia in PBS and wash until only the bone remains. Draw one milliliter of PBS into a syringe and flush the bone marrow cells from one end of the tibia until the bone turns completely white.

Next, collect the cell suspension in a tube. Centrifuge it at 800 G at four degrees Celsius for five minutes. After pipetting out the supernatant, add 500 microliters of red blood cell lysis buffer to the pellet.

After a minute, add one milliliter of separation buffer to terminate the lysis. Centrifuge the suspension again at 800 G at four degrees Celsius for five minutes. Remove the supernatant and place the resulting cell pellet on ice for later use.

For chitosan transfection dissolve four milligrams of chitosan in 20 milliliters of acetic acid solution. Adjust the pH to 5.5 using 10 molar sodium hydroxide. Then dispense 500 microliters of the chitosan solution into individual micro centrifuge tubes.

Now add two, three, four, and five micrograms of ABE plasmids into individual tubes. Dissolve the plasmids in 500 microliters of 30 millimolar sodium sulfate. Then mix 500 microliters of plasmid solution with 500 microliters of chitosan solution in corresponding tubes.

Incubate the tubes in a water bath at 50 to 55 degrees Celsius for 15 minutes. Vortex each tube for 15 to 30 seconds and let them stand undisturbed for 30 minutes. For characterization, analyze the diameter and zeta potential of chitosan using dynamic light scattering.

Maintain the concentration of chitosan at 0.1 milligrams per milliliter in double distilled water. Next, cast 0.1 milligrams per milliliter chitosan samples onto a silicon chip. Add 20 microliters of the resuspended samples onto 200 mesh grids and incubate.

Centrifuge bone marrow cell suspension at 800 G at four degrees Celsius for five minutes. Discard the supernatant, then add 500 microliters of red blood cell lysate. After a minute, add one milliliter of separation buffer to stop the reaction.

Then count the number of cells with a cell counter. Next, transfer 1 million cells into a new micro centrifuge tube. After centrifuging again, pipette out the supernatant and resuspend the cells in 500 microliters of PBS.

Transfer the resuspended cells into a flow cytometry tube and measure transfection efficiency using flow cytometry. For bone marrow cavity injection, fix the anesthetized mouse in a supine position on the operating table. Use adhesive tape to secure the front limb in place.

Disinfect the posterior tibia using an alcohol swab. Then load the plasmid solution into a one milliliter syringe fitted with a 26 gauge needle. Remove any air bubbles and keep the syringe ready for injection.

Next, touch and stabilize the shin of the mouse with your fingers. Position the injection needle to enter the tibial shaft from the tibial plateau near the knee joint. Now rotate the needle parallel to the tibia, insert into the bone marrow cavity and inject slowly over three seconds.

After the injection, slowly withdraw the needle over three seconds. Immediately disinfect the puncture site using an alcohol swab to stop any bleeding. Place the mouse in a warm environment at 37 degrees Celsius to facilitate its recovery.

Nanoparticles formed with the ABE and GRNA plasmids had a uniform spherical morphology and a narrow size distribution with an average size of 202.9 nanometers. A zeta potential of 2.77 millivolts and a poly dispersity index of 0.22. Chitosan embedded ABE plasmid significantly enhanced fluorescent signal in bone marrow cells indicating higher transfection efficiency compared to the control group.

Flow cytometry confirmed that chitosan coded ABE plasmid achieved high transfection efficiency in bone marrow cells. With the peak efficiency of 51.2%observed at a dose of four micrograms, while three micrograms provided a balance of high efficiency of 47.2%Sanger sequencing and PCR of bone marrow cell genomic DNA revealed in vivo gene editing at target site A1 with 14.27%efficiency and at site A2 with 10.69%efficiency.