An enduring challenge for the commercialization of genetic therapies is delivering the therapeutic molecules to the proper destination within the body. Viruses are natural delivery systems for nucleic acids, but they have several significant drawbacks: They aren’t targeted to any one cell type, for instance, and they can’t be re-dosed—the patient develops an immune response that fights the viral vector. They are also expensive and time-consuming to manufacture.
Here we present five companies working, instead, on non-viral delivery methods for gene therapy. Their strategies include lipid nanoparticles, particles combining organic and inorganic components, and electroporation to deliver genetic therapies to the tissues that need them.
Compared with viral delivery vectors, lipid nanoparticles (LNPs) are less likely to trigger an immune response and less expensive to manufacture. Tessera Therapeutics is developing targeted LNPs that can send gene editing RNAs into specific cell types to treat genetic diseases. Their RNA Gene Writer
platform includes an mRNA that encodes the Gene Writer protein plus a template RNA. In the cell, the protein binds to the RNA template and nicks the DNA at a target site in the genome. Then it reverse-transcribes the RNA template into DNA, replacing the disease-causing mutation.
The RNAs are co-formulated with an LNP optimized for delivery to the desired tissue. “Right from the beginning, in addition to the Gene Writing technology, we made an explicit decision to invest in delivery technology as well,” says Hari Pujar, PhD, COO of Tessera. “That has paid off significantly, where we’re now able to deliver our Gene Writing machinery to the liver, to hematopoietic stem cells, and to T cells, allowing us to tackle a variety of diseases.”
Tessera adjusts three different aspects of the LNP to direct it to the desired destination, Pujar says. First, LNPs typically consist of four components, and varying the relative levels of each of those components can influence where the body sends the LNP. Second, they can customize the ionizable lipid component of the particle, which can change how the body deals with them. “We have an extensive discovery engine at Tessera that designs and makes and then screens different ionizable lipids,” Pujar says. “And then lastly, for extra-hepatic targets, we leverage targeting moieties that are attached to the LNP, which further provide cell-based selectivity.”
Preclinical data showed that a single dose of RNA Gene Writer successfully edited the SERPINA1 gene in 79% of hepatocytes. The company is also working on phenylketonuria and sickle cell disease, as well as oncology applications. “We can add a CAR gene to a T cell, but at the same time address another gene,” Pujar says. “What’s beautiful about the Gene Writing technology is that you can do multiplex editing—you can deliver two different cargoes in a single LNP.”
For some therapies, it’s critical not only to get your RNA into the desired cell type, but to avoid uptake by other cells. Generation Bio is developing a T cell-selective LNP to deliver silencing RNA (siRNA) for the treatment of autoimmune disease. The siRNA is intended to reduce the activity of effector T cells without affecting regulatory T cells. The goal for their delivery system is two-fold, explained Geoff McDonough, MD, president and CEO of Generation Bio: keep as much of the dose in circulation as possible, rather than the liver, and selectively target effector T cells.
To address T cell-driven autoimmune diseases, Generation Bio delivers silencing RNA (siRNA) selectively to T cells using specially formulated LNPs designed to avoid the serum-binding proteins that promote unwanted delivery to the liver.
Even with proteins on the surface that target a particular cell type, LNPs typically attract serum-binding proteins, which convey the vast majority to the liver or the spleen. “We’ve engineered a system that defends against that interaction with serum-binding proteins,” McDonough said. “We’ve changed the chemistry, and we’ve also played with the identity of some of the components of that core LNP.” Preclinical data showed that less than 1% of Generation Bio’s LNPs wind up in the liver and spleen, with 99% remaining in circulation and available to T cells. Their LNPs also stay in circulation much longer than conventional LNPs, meaning they have more chances to find their target cells.
Usually, siRNAs are delivered not by LNPs but by creating antibody-siRNA conjugates, but this strategy hasn’t worked in immune cells. This may be because the conjugate can’t escape the endosome, a vesicle that the cell forms to bring molecules from the cell surface to the interior. The endosome eventually matures into a lysosome, which degrades the foreign material. It’s critical for enough of the drug to escape the endosome to accomplish its therapeutic mission before degradation occurs. “For whatever reason, in T cells and other immune cells, that siRNA doesn’t leak out fast enough to work,” McDonough says. “Our LNP is designed to make the endosome open up. When we give siRNA in our LNP system, it comes right out of the endosome and is able to knock down the expression of target proteins in T cells.”
Another approach to nanoparticles combines the advantages of organic and inorganic molecules in a hybrid particle. Suzanne Saffie-Siebert, PhD, founder and CEO of SiSaf, says the idea for Bio-Courier drug delivery technology came out of her background working first with liposomes in graduate school and then developing silicon nanoparticles for drug delivery in a commercial context. “How do you overcome the shortcomings of organic materials, and how do you overcome the challenges of inorganic materials? That was really the idea,” she says.
Saffie-Siebert pointed out several fundamental weaknesses of LNPs, including stability and immunogenicity. For example, traditional LNPs include a component called PEGylated lipid, a polyethylene glycol derivative that helps stabilize the LNP and keeps it in circulation longer. Initially thought to be non-immunogenic, it has since been found to induce production of anti-PEG antibodies. “You have that immunogenicity, but without PEGylated lipid, you have less systemic circulation, and you have a less stable LNP,” said Saffie-Siebert.
SiSaf’s Bio-Courier hybrid nanoparticle incorporates both organic and inorganic components, using a silicon particle in combination with charged lipids and sugar moieties to create a stable, bioabsorbable delivery particle that does not require freezing.
Bio-Courier particles use a hydrolyzable silicon matrix to stabilize the lipid and RNA molecules. This approach eliminates immunogenicity, and it also sidesteps another big drawback of traditional LNPs: the need for cold storage. The hybrid particles can be manufactured empty, transported to their destination, and loaded with nucleic acid payload at the point of care, without freezing. While traditional inorganic drug delivery nanoparticles are not bioabsorbable and may accumulate in the body, risking toxicity, the silicon in Bio-Courier hybrid particles degrades into nontoxic orthosilicic acid.
The hybrid particles can carry different drugs and target different organs around the body. Preclinical data has shown that Bio-Courier particles successfully delivered siRNA to the femur to correct a mutation in the CLCN7 gene, which causes autosomal dominant osteopetrosis type 2 (ADO2), a pediatric disease characterized by excessive bone buildup. “SiSaf is the first company producing a product for the treatment of osteopetrosis,” Saffie-Siebert said. They are working with a U.S. partner to develop two products to correct the mutation that causes corneal dystrophy, transforming growth factor beta I (TGFBI). One uses siRNA to inhibit TGFBI, while the other delivers a CRISPR-Cas9 intended to edit the defective TGFBI gene. These drugs can be delivered topically via eye drops.
PulseSight Therapeutics is advancing a new candidate for age-related macular degeneration (AMD), which affects nearly 20 million people in the U.S. A plasmid encoding a therapeutic protein is introduced into the ciliary muscle cells by a device that delivers precisely calibrated electrical pulses to increase cell permeability. The device also includes an injection guide to ensure the plasmid is delivered to the correct location.
Their lead candidate, PST-611, delivers the gene for transferrin, an iron-chelating protein normally produced in the eye to help maintain iron homeostasis. AMD patients have excess levels of free iron, which is highly toxic to cells. The cells that take up the plasmid begin expressing transferrin, which then moves throughout the eye and binds the excess free iron, protecting retinal cells from damaging effects like ferroptosis, inflammation, and oxidative stress.
PST-611, formerly EYS-611, was originally developed by a company called Eyevensys, and Phase I/II trials done by Eyevensys in both Europe and the U.S. demonstrated the safety and efficacy of the electro-transfection method, using a different plasmid intended to treat chronic non-infectious uveitis. “We know that our administration procedure and our delivery system have a very good safety profile,” says PulseSight CEO Judith Greciet, PharmD. A Phase I trial of PST-611 evaluating safety is expected to begin in summer 2025, with readout expected by early 2026.
After PulseSight Therapeutics’s plasmid is delivered to the ciliary muscle, the cells begin producing transferrin protein, an endogenous iron chelator, which diffuses throughout the vitreous and helps restore healthy levels of free iron.
Preclinical studies have shown protein expression lasting up to six months, but unlike viral gene therapy, the treatment can be reversed if there are any ill effects. “The plasmid’s expression ability fades over time, thus protein expression stops,” says Greciet. “This is a long-duration effect, but it is reversible.”
PST-611 is aimed at treating a form of geographic atrophy (GA), a late-stage form of dry AMD that causes permanent loss of central vision. PulseSight is also developing another gene therapy, PST-809, to treat wet AMD, another advanced form of the disease. This plasmid includes two transgenes: aflibercept, a VEGF inhibitor, and decorin, which has anti-edema and anti-fibrosis activity. Aflibercept (Eylea) is already used to treat wet AMD, delivered by intravitreal injection. The combination, Greciet says, will hopefully boost the efficacy of the treatment.
These new non-virus-based delivery technologies represent the very early stages of a medical revolution, Pujar says. He draws a parallel with antibody-based medicines, which continue to evolve despite having their commercial debut four decades ago. “I’m excited for what this means for the future of genetic medicine,” Pujar says. “Twenty years from now, we’ll be talking about even more sophisticated ways to deliver nucleic acid medicine, and I think it will have a huge impact on human health.”
The post Crossing the Cell Membrane Without Viruses appeared first on GEN - Genetic Engineering and Biotechnology News.
Here we present five companies working, instead, on non-viral delivery methods for gene therapy. Their strategies include lipid nanoparticles, particles combining organic and inorganic components, and electroporation to deliver genetic therapies to the tissues that need them.
Targeted LNPs
Compared with viral delivery vectors, lipid nanoparticles (LNPs) are less likely to trigger an immune response and less expensive to manufacture. Tessera Therapeutics is developing targeted LNPs that can send gene editing RNAs into specific cell types to treat genetic diseases. Their RNA Gene Writer
The RNAs are co-formulated with an LNP optimized for delivery to the desired tissue. “Right from the beginning, in addition to the Gene Writing technology, we made an explicit decision to invest in delivery technology as well,” says Hari Pujar, PhD, COO of Tessera. “That has paid off significantly, where we’re now able to deliver our Gene Writing machinery to the liver, to hematopoietic stem cells, and to T cells, allowing us to tackle a variety of diseases.”
Tessera adjusts three different aspects of the LNP to direct it to the desired destination, Pujar says. First, LNPs typically consist of four components, and varying the relative levels of each of those components can influence where the body sends the LNP. Second, they can customize the ionizable lipid component of the particle, which can change how the body deals with them. “We have an extensive discovery engine at Tessera that designs and makes and then screens different ionizable lipids,” Pujar says. “And then lastly, for extra-hepatic targets, we leverage targeting moieties that are attached to the LNP, which further provide cell-based selectivity.”
Preclinical data showed that a single dose of RNA Gene Writer successfully edited the SERPINA1 gene in 79% of hepatocytes. The company is also working on phenylketonuria and sickle cell disease, as well as oncology applications. “We can add a CAR gene to a T cell, but at the same time address another gene,” Pujar says. “What’s beautiful about the Gene Writing technology is that you can do multiplex editing—you can deliver two different cargoes in a single LNP.”
Stay on target
For some therapies, it’s critical not only to get your RNA into the desired cell type, but to avoid uptake by other cells. Generation Bio is developing a T cell-selective LNP to deliver silencing RNA (siRNA) for the treatment of autoimmune disease. The siRNA is intended to reduce the activity of effector T cells without affecting regulatory T cells. The goal for their delivery system is two-fold, explained Geoff McDonough, MD, president and CEO of Generation Bio: keep as much of the dose in circulation as possible, rather than the liver, and selectively target effector T cells.
To address T cell-driven autoimmune diseases, Generation Bio delivers silencing RNA (siRNA) selectively to T cells using specially formulated LNPs designed to avoid the serum-binding proteins that promote unwanted delivery to the liver.
Even with proteins on the surface that target a particular cell type, LNPs typically attract serum-binding proteins, which convey the vast majority to the liver or the spleen. “We’ve engineered a system that defends against that interaction with serum-binding proteins,” McDonough said. “We’ve changed the chemistry, and we’ve also played with the identity of some of the components of that core LNP.” Preclinical data showed that less than 1% of Generation Bio’s LNPs wind up in the liver and spleen, with 99% remaining in circulation and available to T cells. Their LNPs also stay in circulation much longer than conventional LNPs, meaning they have more chances to find their target cells.
Usually, siRNAs are delivered not by LNPs but by creating antibody-siRNA conjugates, but this strategy hasn’t worked in immune cells. This may be because the conjugate can’t escape the endosome, a vesicle that the cell forms to bring molecules from the cell surface to the interior. The endosome eventually matures into a lysosome, which degrades the foreign material. It’s critical for enough of the drug to escape the endosome to accomplish its therapeutic mission before degradation occurs. “For whatever reason, in T cells and other immune cells, that siRNA doesn’t leak out fast enough to work,” McDonough says. “Our LNP is designed to make the endosome open up. When we give siRNA in our LNP system, it comes right out of the endosome and is able to knock down the expression of target proteins in T cells.”
Best of both worlds
Another approach to nanoparticles combines the advantages of organic and inorganic molecules in a hybrid particle. Suzanne Saffie-Siebert, PhD, founder and CEO of SiSaf, says the idea for Bio-Courier drug delivery technology came out of her background working first with liposomes in graduate school and then developing silicon nanoparticles for drug delivery in a commercial context. “How do you overcome the shortcomings of organic materials, and how do you overcome the challenges of inorganic materials? That was really the idea,” she says.
Saffie-Siebert pointed out several fundamental weaknesses of LNPs, including stability and immunogenicity. For example, traditional LNPs include a component called PEGylated lipid, a polyethylene glycol derivative that helps stabilize the LNP and keeps it in circulation longer. Initially thought to be non-immunogenic, it has since been found to induce production of anti-PEG antibodies. “You have that immunogenicity, but without PEGylated lipid, you have less systemic circulation, and you have a less stable LNP,” said Saffie-Siebert.
SiSaf’s Bio-Courier hybrid nanoparticle incorporates both organic and inorganic components, using a silicon particle in combination with charged lipids and sugar moieties to create a stable, bioabsorbable delivery particle that does not require freezing.
Bio-Courier particles use a hydrolyzable silicon matrix to stabilize the lipid and RNA molecules. This approach eliminates immunogenicity, and it also sidesteps another big drawback of traditional LNPs: the need for cold storage. The hybrid particles can be manufactured empty, transported to their destination, and loaded with nucleic acid payload at the point of care, without freezing. While traditional inorganic drug delivery nanoparticles are not bioabsorbable and may accumulate in the body, risking toxicity, the silicon in Bio-Courier hybrid particles degrades into nontoxic orthosilicic acid.
The hybrid particles can carry different drugs and target different organs around the body. Preclinical data has shown that Bio-Courier particles successfully delivered siRNA to the femur to correct a mutation in the CLCN7 gene, which causes autosomal dominant osteopetrosis type 2 (ADO2), a pediatric disease characterized by excessive bone buildup. “SiSaf is the first company producing a product for the treatment of osteopetrosis,” Saffie-Siebert said. They are working with a U.S. partner to develop two products to correct the mutation that causes corneal dystrophy, transforming growth factor beta I (TGFBI). One uses siRNA to inhibit TGFBI, while the other delivers a CRISPR-Cas9 intended to edit the defective TGFBI gene. These drugs can be delivered topically via eye drops.
The eyes have it
PulseSight Therapeutics is advancing a new candidate for age-related macular degeneration (AMD), which affects nearly 20 million people in the U.S. A plasmid encoding a therapeutic protein is introduced into the ciliary muscle cells by a device that delivers precisely calibrated electrical pulses to increase cell permeability. The device also includes an injection guide to ensure the plasmid is delivered to the correct location.
Their lead candidate, PST-611, delivers the gene for transferrin, an iron-chelating protein normally produced in the eye to help maintain iron homeostasis. AMD patients have excess levels of free iron, which is highly toxic to cells. The cells that take up the plasmid begin expressing transferrin, which then moves throughout the eye and binds the excess free iron, protecting retinal cells from damaging effects like ferroptosis, inflammation, and oxidative stress.
PST-611, formerly EYS-611, was originally developed by a company called Eyevensys, and Phase I/II trials done by Eyevensys in both Europe and the U.S. demonstrated the safety and efficacy of the electro-transfection method, using a different plasmid intended to treat chronic non-infectious uveitis. “We know that our administration procedure and our delivery system have a very good safety profile,” says PulseSight CEO Judith Greciet, PharmD. A Phase I trial of PST-611 evaluating safety is expected to begin in summer 2025, with readout expected by early 2026.
After PulseSight Therapeutics’s plasmid is delivered to the ciliary muscle, the cells begin producing transferrin protein, an endogenous iron chelator, which diffuses throughout the vitreous and helps restore healthy levels of free iron.
Preclinical studies have shown protein expression lasting up to six months, but unlike viral gene therapy, the treatment can be reversed if there are any ill effects. “The plasmid’s expression ability fades over time, thus protein expression stops,” says Greciet. “This is a long-duration effect, but it is reversible.”
PST-611 is aimed at treating a form of geographic atrophy (GA), a late-stage form of dry AMD that causes permanent loss of central vision. PulseSight is also developing another gene therapy, PST-809, to treat wet AMD, another advanced form of the disease. This plasmid includes two transgenes: aflibercept, a VEGF inhibitor, and decorin, which has anti-edema and anti-fibrosis activity. Aflibercept (Eylea) is already used to treat wet AMD, delivered by intravitreal injection. The combination, Greciet says, will hopefully boost the efficacy of the treatment.
The future of genetic medicine
These new non-virus-based delivery technologies represent the very early stages of a medical revolution, Pujar says. He draws a parallel with antibody-based medicines, which continue to evolve despite having their commercial debut four decades ago. “I’m excited for what this means for the future of genetic medicine,” Pujar says. “Twenty years from now, we’ll be talking about even more sophisticated ways to deliver nucleic acid medicine, and I think it will have a huge impact on human health.”
The post Crossing the Cell Membrane Without Viruses appeared first on GEN - Genetic Engineering and Biotechnology News.