In Vivo RNA Injection: A New Treatment for Cancer, Autoimmune Disease and Gene Therapy

(Posted on Friday, June 14, 2024)

​​Over the last thirty years, protein-based injections and DNA therapeutics accounted for the majority of newly introduced treatments. However, these medicines remain out of reach for many around the world due to high pricing, with some therapies ranging from thousands to several millions of dollars to purchase. 

A new medical era may put these concerns to rest by ushering in a wave of more effective and less expensive treatments. RNA-based therapeutics, also called RNA direct injections or infusions, lie at the heart of this revolution. In vivo injections of RNA, whether in vaccines, drugs, or novel therapies, embrace a new approach to treating disease: targeting and modifying cells within the body. Harnessing this cost-effective, adaptable technology promises to extend treatment accessibility for all. 

This article will delve into the historical evolution of RNA technology, illuminate how RNA direct injections are reshaping the medical landscape today, and examine the potential applications and pitfalls of this platform as the field progresses. 

Shifting From DNA to RNA 

The roots of this new era germinated in the late 1970s and 1980s with the DNA revolution. During this time, scientists sought to understand DNA and its role in biology and medicine. These investigations led to breakthroughs in drug development.  

Protein and Antibody Therapeutics

DNA discoveries such as recombinant DNA technology brought us protein and antibody therapeutics to treat heart disease, obesity, cancer and more. The technology allows the production of genetically engineered proteins and therapeutic agents by isolating and expressing the DNA that encodes them. 

Proteins such as antibodies, enzymes, and hormones are now used in medicines to stimulate the immune system, replace protein deficiency, or target specific biological processes such as cancer or autoimmunity treatments. Recognizable protein-based drugs and vaccines include insulin, growth factors, the hepatitis B vaccine, antibodies for autoimmune disease treatment, and more. Protein-based therapeutics dominate other products in global biopharmaceutical sales volume, totaling over $270 billion in international sales. Synthetically-made antibodies comprised around eighty percent of total protein-based biopharma sales between 2018 and 2022.  

Although effective, protein-based treatments are expensive to manufacture and purchase. Some products, such as cancer treatment rituximab, sell for billions of dollars per kilogram. Much of the expense arises from manufacturing costs. Cultures of mammalian cells or bacteria are carefully cultivated to produce select proteins. Then, the proteins must be meticulously separated from the culture to achieve a pure, therapeutically viable end product.  

Gene Therapy 

Advances in DNA also propelled gene therapy research. The ability to clone genes, synthesize DNA sequences, and manipulate genetic material proved invaluable to this budding field. Gene therapies work either by adding functional genetic material to compensate for faulty genes or directly modifying a person’s DNA to correct genetic abnormalities. These therapeutics treat diseases at their genetic core by changing the instructions for protein production.

Notably, only a minority of gene therapies today rely on DNA alone. The US has approved 17 gene therapies; almost all these treatments are prohibitively expensive. Gene therapies cost around $1.5 million per treatment, but many exceed this estimate by millions. The most costly gene therapy to date is Lenmeldy, a recently approved treatment for people with a rare, inherited motor neuron disease; it sells for $4.25 million in the United States. 

 Companies argue that the price tag is justified given the complicated manufacturing and lifetime benefits provided, especially for rare diseases. Yet, left as is, people have few options. Some buy the therapy despite the exorbitant price; the majority suffer without it.  

Cell Therapy for Regenerative Medicine 

Our understanding of stem cell transplants and other cell therapies has blossomed within the last twenty years. With these treatments, the unique properties of self-renewing stem cells and cancer-killing immune cells are harnessed to regenerate damaged cells and tissues in the human body or replace them with healthy and fully functional cells. 

These treatments extensively alter live cells to treat disease. The cells can be genetically modified, as seen in cancer-killing CAR T therapy. The cells are then cultivated in large numbers in a lab before being returned to the patient. Since the cells originate from the same individual receiving the treatment, the patient is less likely to experience graft rejection or immune reactions. 

A limitation of these cell-based therapeutics is the use of the patient’s own cells. Using a patient’s cells avoids the famous self-versus-non-self rejection problem, where the immune system mistakes the new cells as foreign or harmful and attacks them. However, additional chemotherapy pretreatment is often necessary to ensure engraftment of the modified cells—a risky process at best. 

Each treatment is tailored to the individual. This technically demanding, resource-intensive process depends on highly skilled staff, cutting-edge technology, and specialized laboratories. These factors translate to an expensive product that cannot be scaled and can take weeks to produce. For context, cell therapies can average around $1 million per treatment. 

Some researchers have turned to allogeneic therapies to overcome cell-based production dilemmas. These “off-the-shelf” therapies rely on donor cells instead of patient cells. Although allogeneic products can be produced in bulk and used for many, they risk graft rejection.

RNA Technology Overcomes Graft-vs-Host and Host-vs-Graft Rejection

There is a pressing need to decrease manufacturing expenses and improve access to these lifesaving drugs. The newest inflection point in the revolution may address these concerns: Ribonucleic acid (RNA) technology. RNA is a type of genetic material that instructs protein assembly. These molecules target various biological processes and perform essential functions in DNA, other RNAs and proteins. In vivo injections and infusions of RNA rely on standardized and scalable processes, which reduces manufacturing costs and complexity. 

Vaccines

The success of mRNA vaccines during the COVID-19 pandemic demonstrated the power of RNA in vivo injections. The Pfizer/BioNTech and Moderna vaccines are linear messenger RNAs. The RNA is surrounded by a protective lipid layer and injected directly into the muscle. The messenger RNA (mRNA) enters cells and is translated into a viral protein recognized by the immune system. Billions of these vaccines were produced within a few months at a fraction of the cost of traditional vaccines—for less than $3 per dose, according to the Royal Society of Medicine. 

Companies worldwide are now racing to create similar vaccines to protect us from infections of influenza, respiratory syncytial virus (RSV), dengue, tuberculosis, filoviruses such as Ebola and Lassa, and many other dangerous pathogens. These, too, will cost a few dollars to produce in bulk.

RNA Interference

A new class of RNA drugs called RNA interference actually preceded the two COVID RNA vaccines. These drugs contain small bits of RNA, usually either microRNAs or interfering RNAs. These small RNAs do not make proteins. Instead, they bind to specific messenger RNAs (mRNAs) and trigger their destruction. Such RNAs eliminate or reduce the amount of protein that causes disease made by the mRNA. Like RNA vaccines, the small RNA is surrounded by a lipid membrane for delivery or coupled to the sugar molecule GalNAc (N-Acetylgalactosamine) that binds to a receptor on liver cells. As of this writing, the FDA has approved seven such drugs by US biotechnology company Alnylam.  

Neither the current vaccines nor RNA interference drugs target specific cells (except for GalNAc-modified RNA). The lipid-wrapped RNA is taken up nonspecifically by immune cells in the lymph nodes, and the silencing RNA modifies liver proteins. The lipid nanoparticle drugs enter through large, window-like pores in the blood vessels called fenestrations that bring nutrients from the intestines to the liver. 

Without the ability to target specific cells with injection or infusion, RNA technology will be restricted to use in vaccines or a small set of diseases that do not require the RNA to enter a cell of choice. 

One method to overcome this difficulty is to apply RNA drugs to modify specific types of cells grown outside the body, usually in culture dishes. Typically, such cells are extracted from a patient, purified, exposed to the RNA drug, selected for the desired property, expanded, and infused back into the patient. The modified cells might even be highly specific cells developed by manipulating embryonic-type cells created from the patient’s blood or skin.

There are several limitations to treating cells in culture. The most serious is relying on the patient’s cells to avoid rejection. Attempts to reduce rejection by manipulating modified donor cells have yielded minimal success. Another serious disadvantage of this method is the expense and time required to treat single individuals, limiting such treatment to the very few.  

RNA Gene Therapy

RNA-based drugs can also modify or place entire genes. The first such US FDA-approved drug wields RNA-directed DNA change to activate a fetal hemoglobin gene to treat two inherited blood diseases: sickle cell disease and thalassemia. In this case, blood-forming cells are harvested and purified from a person with inherited disease, modified using RNA-directed CRISPR/Cas9 gene editing tools, selected for production of functional hemoglobin, expanded, and infused back into the patient after treatment of the patient with potent immunosuppressant to ensure engraftment of the modified cells.

 The ability to edit, remove, and replace genes in cultured cells is improving rapidly, opening the possibility of treating many inherited and even gene-based acquired diseases. However, such therapies have severe cost limitations from modifying the patient’s cells in culture. 

The Solution: In Vivo RNA Injections & Infusions  

Targeting RNA Drugs

The solution to both the issue of immune rejection and the cost of manufacturing new medicines is the direct injection of RNA-based drugs. Rather than manipulate the cells externally, we can borrow the body’s natural ability to read RNA and produce proteins. 

This method targets specific cells in an intact body by coupling GalNAc to the injected RNA and carrying it directly to liver cells. After the particles enter the liver’s pores, the liver can produce the missing protein. 

However, not all treatments have such a simple solution. Many require that the RNA be modified to a specific cell type. For example, hemoglobin defects must be remedied by altering a blood cell precursor. Moreover, some RNA drugs may cause adverse effects upon entering an “off-target” cell.

 Use of Antibodies to Target Cell Receptors

Precise targeting is crucial in attaining a highly effective mRNA product. The RNA carrier should only change a specific subset of cells to maximize their therapeutic effect. 

Antibodies can provide this missing specificity. When attached to a vector surface, antibodies bind to selective protein markers on other cells called antigens. As a result, the drug only interacts with targeted cells. 

On Phospholipid-Based Carriers 

Lipid-based carriers, including liposomes or lipid nanoparticles, can achieve targeted delivery with antibodies conjugated to their surface. One preclinical study of heart damage demonstrates effective targeting by covering lipid nanoparticle vehicles with antibodies that target CD5 antigens on immune T cells. This approach successfully delivers the therapeutic mRNA and temporarily transforms T cells against fibrosis, a type of scarring. Mice with fibrotic hearts receive this infusion and experience improved heart function and reduced scarring.  

Researchers at the University of Pennsylvania employ a similar tactic to address blood stem cells. Here, the authors decorate the lipid nanoparticle surface with antibodies against an antigen commonly found on blood stem cells: antigen CD117. The effect of this carrier changes depending on the internal cargo. The vector can correct sickle cell disease in blood stem cells in culture if gene-editing mRNA is placed inside the carrier. Alternatively, this system can also carry mRNA called p53 up-regulated modulator of apoptosis, or PUMA, to blood stem cells. This nanoparticle-mRNA combo selectively depletes blood stem cells in mice. The process mimics preconditioning chemotherapy required for stem cell transplantations but without the toxic effects.

On the surface of virus particles 

Antibodies can also enhance viral vectors. In these vectors, the pathogenic material inside viruses such as adenoviruses, retroviruses, or lentiviruses is replaced with chosen mRNA sequences. The viral vector delivers the therapeutic cargo instead of infecting the target cell. 

One paper published in Nature Biotechnology yields preclinical success with enveloped delivery systems, a type of heavily customized viral vector infusion. The authors gut an HIV of its pathogenic genes and fuse gene-editing Cas9 proteins to the viral structure. To aid targeting and vehicle entry into cells, the outside of the vehicle is covered with antibody fragments called single-chain variable fragments (scFVs), which bind readily to white blood T cells, and VSVGmut, a glycoprotein that aids viral vector entry into the cell.

The enveloped delivery system can haul artificial genes, CRISPR-based constructs, or both. In the experiment, the infusion targets T cells in mice to mimic Chimeric Antigen Receptor T cell Therapy (CAR T therapy). The goal is to convert normal white blood cells in the body into CAR T cells without cell extraction. 

The vehicles carried transgenes encoding a CD19 CAR receptor. The CRISPR-cas9 proteins integrated into the vehicle are programmed to turn off T cell receptor alpha constant (TRAC) genes on the T cells, encouraging the immune system to accept the newly formed CAR T cells. 

Immunodeficient mice treated with this infusion show signs of gene editing. A portion of T cells sport the synthetic receptor and demonstrate modified alleles. The injection was not detected in the liver, suggesting that this design successfully avoids liver toxicity. Likely, it is possible to modify adenoviruses, adeno-associated viruses and other common viral vectors in the same manner. 

Antibodies Directly Coupled to RNA

Although not as ventured, it is possible to directly couple antibodies to RNA instead of a carrier. One study demonstrates proof-of-concept with small interfering RNA (siRNA). The entire complex relies on three joined components: 1) a small, positively charged protein called protamine; 2) a crosslinking agent; and 3) an antibody against epidermal growth factor or EGFR on tumor cells. Electrostatic interactions allow the negatively charged siRNA to join the complex at the protamine end. 

This system can carry siRNA to tumor cells in culture and silence select genes. The construct could be adapted to target different cells by interchanging the desired antibody. Another benefit is the potential to save time and resources, as each component is readily available in its purified form and does not need to be engineered specifically for the task. 

Direct Attachment of a Targeting Molecule RNA 

Another option to improve specificity is to attach a targeting molecule directly to the RNA rather than the vector. Alnylam researchers accomplish this by conjugating GalNAc to small interfering RNA. The synthetic GalNAc delivers the RNA to liver cells, where it can silence genes and reduce harmful protein numbers. The team modified first-generation designs to prevent the drug from targeting cells other than the liver. The change significantly improves the therapeutic window in rats with siRNAs that have established hepatotoxicity driven by off-target effects. Their redesigned treatment for chronic hepatitis B (HBV) infection shows improved specificity and has entered clinical development. 

Site-Specific In Vivo Injection

How an RNA drug is introduced to the body can significantly impact its precision. Unlike systemic infusions that travel the body, site-specific in vivo injections localize the product’s therapeutic effect. This approach benefits sites such as the brain or eye, where a specific cell type is not crucial.  

For example, one clinical trial turns to intrathecal injections to deliver their RNA-interfering product for Alzheimer’s disease. The drug is injected into the back, in the space just outside the spinal cord. The injection bypasses the blood-brain barrier, a network of blood vessels and tissues that selectively filters what enters the brain, and delivers the product directly to the central nervous system. Phase I results suggest that their treatment is well-tolerated; it successfully reduces amyloid precursor proteins (APP) in the cerebrospinal fluid—almost up to 90% at the highest dose tested. 

Combating Roadblocks 

Although in vivo injections and infusions of RNA are promising research avenues, they are accompanied by pitfalls. Further investigation is necessary to optimize each system.  

Injections with viral vectors will need to overcome manufacturing challenges. Similar to producing cell therapies, cultivating viral vectors is complex and demanding. Live host cells are transfected with the viral vectors and expanded to great numbers; then, the vectors must be harvested, purified and stored until use. Another consideration is the high demand for viral vectors in gene therapy research. As witnessed during the pandemic, vector shortages could arise again if manufacturers do not grow capacity accordingly.  

Safety may also be a concern. Viral vectors can trigger unwanted immune system reactions. These vehicles can also integrate viral DNA into the host cell genome, leading to DNA mutations, cancer, or other harmful effects. However, newer generations of viral vectors have a lower propensity for genome integration. 

Liposomes and lipid nanoparticles are comparatively easier to mass-produce than their viral counterparts. However, these vectors can face stability issues and do not enter cells as efficiently as viral vectors. Changes will be necessary to evoke a lasting expression of desired genes.

Bright Future Horizons 

The field of medicine is transforming. It has witnessed significant strides in recent years with the advent of vaccines and personalized therapies. However, many advances are limited by their accessibility, cost, and potential risks. In this landscape, mRNA injections emerge as a compelling solution by delivering genetic material directly to cells within the body. This method could simplify manufacturing processes, address safety concerns, and slash prices for people who need these therapeutics. With additional research on precise targeting and optimization, this approach could inject hope for a world where medicines are affordable and accessible for all. 

Read the original article on Inside Precision Medicine.