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Gene Theraphy Series
Gene therapy: nanotechnology as an ally in the treatment of various skin diseases
NanoGeneSkin Laboratory in Ribeirão Preto focuses efforts on developing research to treat, primarily, skin cancer, vitiligo, and psoriasis
Ilustration: kjpargeter/Freepik
Recently, USP’s Ribeirão Preto Blood Center requested authorization from the Brazilian Health Surveillance Agency (Anvisa) and began phase 2 clinical trials in individuals with B-cell acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma. Data from the first stage of the CAR-T cell research, conducted with four patients from the Ribeirão Preto Clinical Hospital (HCRP), confirmed that the treatment is safe.
CAR-T cell therapy is one of several types of cell and gene therapy under study in Brazil and worldwide. It consists of manipulating (or reprogramming) the patient’s own immune system cells (T lymphocytes) so they recognize and attack tumor cells. In the therapy developed by the Blood Center, CAR-T cells were trained to fight the tumor by recognizing a specific target: the CD19 antigen, present only in B-cell acute lymphoblastic leukemia and B-cell non-Hodgkin lymphoma cells.
Since the first human gene therapy protocol in 1990, much has happened. Today, there is great hope that this approach can combat diseases that still have no cure, such as cancer. A quick search on clinicaltrials.gov – an online database of clinical research studies in the United States – shows that about 400 clinical studies involving gene therapy and cancer are currently underway.
Jornal da USP now presents some initiatives within and outside USP that aim to treat different diseases through gene manipulation.
Turning Off Genes
At the NanoGeneSkin Laboratory, research focuses on the use of nanotechnology to treat various skin diseases, such as psoriasis, skin cancer, and vitiligo. Coordinated by Maria Vitória Bentley, professor at the Ribeirão Preto School of Pharmaceutical Sciences (FCFRP-USP) and coordinator of the National Institute of Science and Technology in Pharmaceutical Nanotechnology (INCT-INOFAR), the group’s most recent research line involves developing treatments based on gene silencing technology.
In the treatment, scientists study the genes that are overproduced in certain pathological processes and silence them, that is, prevent them from being expressed. “In an inflammatory process, the genes responsible for producing certain pro-inflammatory cytokines – important for various biological processes – are overexpressed, leading to the manifestation of the disease,” explains Maria Vitória Bentley. “Our proposal is to administer the gene that will silence the production of these substances responsible for the pathological process.”
Bentley notes that this therapy differs from DNA gene therapy, which enters the cell nucleus to produce a specific gene. This technique has been used in some vaccines, for example.
Maria Vitória Lopes Badra Bentley – Jornal da USP
In antisense therapy (siRNA), as it is also called, the goal is to silence deregulated genes that interrupt or alter the structure of proteins, causing them to malfunction. “Psoriasis and skin cancer are examples of diseases in which genes are overexpressed,” the researcher explains.
Genetic Factors
Psoriasis is a chronic inflammatory skin condition, characterized by the formation of white or pink patches that later peel and turn whitish.
Its causes are not yet fully understood, but studies suggest a link to genetic factors. Some theories indicate that the disease develops when T lymphocytes (the body’s defense cells) release inflammatory substances that trigger the reddish appearance of the patches. The body then responds to this inflammation, and the presence of another type of defense cell, neutrophils, leads to skin peeling.
“We discovered that the main pro-inflammatory cytokine being overproduced is TNF-alpha,” explains Maria Vitoria. “We identified a silencer, which we call siRNA TNF-alpha.”
The biggest challenge, however, was still ahead. The outermost layer of the skin, the stratum corneum, acts as a barrier against microorganisms, dehydration, and drug permeation – the passage of fluids through skin layers. “This silencer would have to enter, break through the stratum corneum, and reach the layers where the disease develops,” the researcher reports.
At the same time, Bentley’s group was developing a type of liquid crystal nanoparticle, which they later named soft nanoparticles. Unlike solid nanoparticles, liquid crystal nanoparticles are composed of lipid-based crystalline gel in a hexagonal arrangement. “This type of lipid-based liquid crystal interacts with the stratum corneum and alters its permeability,” the professor highlights.
A topical treatment for psoriasis using nanotechnology and antisense therapy is the proposal that Bentley’s group has been investigating for 15 years.
From Bench to Animal Experiments
Initially, the group conducted experiments on cell cultures (inflamed skin membranes) and observed a positive effect when treated with soft nanoparticles containing siRNA TNF-alpha. “We treated the cells and checked for silencing of TNF-alpha production,” Bentley explains.
The next step involved trials in animal models, using hairless mice. Scientists irritated the animals’ skin to verify whether cytokine production would increase, and indeed, the inflammation led to high TNF-alpha levels. “Then we applied the nanoparticles with the silencing gene to this inflamed skin,” she notes. Different protocols were tested, with variations in duration and number of applications. Afterwards, the animals were sacrificed and the skin was removed for analysis.
The results, published in the Journal of Controlled Release, were encouraging. “We observed that when siRNA TNF-alpha is incorporated into the liquid crystal nanoparticle, there is greater silencing of the production of this pro-inflammatory cytokine, reaching baseline levels of a non-diseased cell.”
Skin Cancer
“The issue of melanoma is very complex and requires advanced therapeutic strategies for its cure,” explains Maria Vitoria Bentley. Melanoma is the most severe type of skin cancer (representing only 4% of malignant neoplasms of the organ) due to its high potential to metastasize, that is, to spread to other organs.
Melanoma originates in melanocytes (cells that produce melanin, the pigment that gives skin its color) and can appear anywhere on the body as spots, moles, or marks. According to the professor, “gene silencing therapy is important in this process because, in this disease, several genes are deregulated, including those that cause resistance to chemotherapy.”
The topical treatment approach under development may support therapy when cancer is in its early stages. The nanoparticle created by the group incorporates a chemotherapeutic agent together with gene silencers that are overexpressed in the disease.
“We can use gene silencers that block the apoptosis process [programmed cell death], which induces cancer cell proliferation. Thus, we enhance the effect of the chemotherapeutic agent and make the treatment more effective.”
The article published in the European Journal of Pharmaceutical Sciences details the entire development process of the nanoparticle, as well as skin penetration tests conducted in vitro in 3D cell cultures (spheroids mimicking small tumors) and tumor regression evaluation.
Vitiligo is another condition targeted by NanoGeneSkin. In a 2008 publication, the group developed a nanoparticle containing siRNA that silences the production of a protein located on the melanocyte membrane. This protein attracts autoantibodies that destroy melanocytes. “Our in vitro studies showed that we were able to silence 80% of the production of this protein,” Bentley points out. “Since vitiligo is a complex and multifactorial disease, we are currently studying the effect of silencing other genes that are overexpressed in the disease.”
Future
The group’s next challenge is to develop an intranasal messenger RNA vaccine delivered via nanoparticles. The idea came after the COVID-19 pandemic, while scientists around the world worked to develop, in record time, a vaccine capable of curbing the spread of SARS-CoV-2 globally.
“We thought: we have a delivery system, a differentiated nanoparticle, which has the potential to interact with the nasal mucosa, we have a proof-of-concept model, but we still don’t have a specific mRNA to develop the vaccine,” highlights Maria Vitoria Bentley.
“We are now working on pharmaceutical development, verifying physicochemical aspects of interaction with the nasal mucosa, functionality tests in vitro and in animal models,” she says, emphasizing that the key point of the nanoparticle is its ability to interact with biomembranes.
“We want to see the population, the patient, benefiting from what we set as our scientific goal,” summarizes the INCT coordinator.
More information: vbentley@usp.br, with Maria Vitoria Bentley
English version: Nexus Traduções, edited by Denis Pacheco
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A reprodução de matérias e fotografias é livre mediante a citação do Jornal da USP e do autor. No caso dos arquivos de áudio, deverão constar dos créditos a Rádio USP e, em sendo explicitados, os autores. Para uso de arquivos de vídeo, esses créditos deverão mencionar a TV USP e, caso estejam explicitados, os autores. Fotos devem ser creditadas como USP Imagens e o nome do fotógrafo.
A reprodução de matérias e fotografias é livre mediante a citação do Jornal da USP e do autor. No caso dos arquivos de áudio, deverão constar dos créditos a Rádio USP e, em sendo explicitados, os autores. Para uso de arquivos de vídeo, esses créditos deverão mencionar a TV USP e, caso estejam explicitados, os autores. Fotos devem ser creditadas como USP Imagens e o nome do fotógrafo.