Terapia génica lentiviral en ratones SCA3: Seguridad a largo plazo

Escrito por la Dra. Ambika Tewari Editado por la Dra. Hayley McLoughlin. Publicado inicialmente en el 6 de agosto de 2021. Traducción al español fueron hechas por FEDAES.

La expresión lentiviral de un ARNhc contra ataxina-3 fue bien tolerada y no produjo efectos adversos medibles en ratones de tipo salvaje.

La evaluación del perfil de seguridad es un paso necesario y crucial para calificar una terapia para su uso en pacientes. La terapia génica es una técnica experimental que ha demostrado un enorme progreso en el tratamiento o la reversión de una enfermedad, específicamente los trastornos monogénicos.

Es importante investigar con detenimiento la seguridad y la tolerancia de la terapia génica para evaluar su idoneidad para los ensayos clínicos. Las herramientas de terapia génica se pueden utilizar de diferentes maneras para lograr el mismo efecto terapéutico: el gen defectuoso se puede reemplazar con una copia sana, el gen mutado se puede reparar o la copia mutante del gen se puede silenciar.

La ataxia espinocerebelosa tipo 3 (SCA3) o enfermedad de Machado-Joseph (MJD) causa una pérdida progresiva de neuronas en la médula espinal y en varias regiones del cerebro. Esto incluye el cerebelo, tronco encefálico, cuerpo estriado y sustancia negra. Estas neuronas tienen funciones cruciales. Sin estas neuronas, los pacientes experimentan descoordinación motora, pérdida del equilibrio y, en casos graves, muerte prematura.

Si bien se sigue logrando un gran progreso en la comprensión de cómo una mutación en un solo gen, Ataxin-3, causa los síntomas de SCA3, todavía no existe un tratamiento para detener la progresión de la enfermedad. Como trastorno monogénico, SCA3, al igual que otras ataxias espinocerebelosas (SCA), es un candidato prometedor para la terapia génica. Si bien aún no existen terapias génicas aprobadas para la SCA, existen varios laboratorios de investigación y empresas que trabajan para lograr este objetivo.

An artist's drawing of scientists standing infront of a giant piece of DNA and drugs
Este es un momento verdaderamente emocionante para la terapia génica, pero también es importante mantener la seguridad de los pacientes como una prioridad absoluta. Foto utilizada bajo licencia por Visual Generation / Shutterstock.com .

Los investigadores de este estudio han estado trabajando en la terapia génica para SCA3 desde 2008. Han investigado cómo la terapia génica podría ofrecer protección contra una mayor disminución, en varios modelos de células y ratones de SCA3. Utilizaron un enfoque en el que disminuyeron los niveles del gen Ataxin-3 mutante mientras dejaban intacto el gen Ataxin-3 normal. Esto se conoce como focalización específica de alelos. Demostraron que utilizando esta técnica, podrían reducir significativamente los cambios de comportamiento y neuropatológicos que ocurren en ratones SCA3. Los ratones tratados con la terapia génica mostraron mejoras en su equilibrio y coordinación motora.

La terapia génica en su forma más básica involucra dos componentes, el gen que reemplazará o eliminará el gen enfermo y un vector que transportará este nuevo gen a su sitio de acción. Los vectores más comúnmente utilizados en la actualidad son los virus adenoasociados (AAV) seguidos por los retrovirus. Estos virus se han diseñado específicamente para llevar a su pasajero a la ubicación especificada. Si bien ambos vectores han pasado por varios años de pruebas preclínicas y clínicas para numerosos candidatos a terapia génica, quedan preguntas sobre su seguridad. (1) ¿El producto de terapia génica continúa expresándose en el área objetivo a largo plazo? (2) Si hay expresión a largo plazo, ¿causa algún efecto adverso mensurable en el área objetivo? (3) ¿La expresión a largo plazo afecta el funcionamiento normal de las células / órganos diana?

En este estudio actual, los investigadores probaron sistemáticamente la seguridad de reducir los niveles de Ataxin-3 mutante específicamente en el cuerpo estriado de ratones adultos de tipo salvaje. El gen, un ARN en horquilla corto (ARNhc) que reduce el nivel de la proteína ataxina-3 mutante, se empaquetó en un vector. El vector utilizado en este estudio es un lentivirus. Este es un tipo de retrovirus que infecta las células que no se dividen, como las neuronas del cerebro. Por lo tanto, cuando el vector lentivirus se inyecta en el cerebro, transporta su carga de ARNhc a las neuronas.

Como control experimental, este estudio utilizó tanto ratones que no fueron inyectados como ratones inyectados con una sustancia inerte en las mismas dos ubicaciones que el shRNA. Dado que los ratones de tipo salvaje no expresan ataxina-3 mutada, este estudio solo analiza los efectos a largo plazo de expresar el lentivirus con el ARNhc.

En tres puntos de tiempo diferentes (2, 8 y 20 semanas después de la inyección o administración del vector y su carga de ARNhc) se sacrificaron los ratones. Luego, se recolectaron y analizaron sus cerebros. Una característica importante de cualquier producto de terapia génica es su perfil de expresión. Esto incluye información como su distribución tisular hasta la duración de su expresión. El ARNhc contra la ataxina-3 mutante contenía un informador. Este informe permitiría identificar cualquier célula con ARNhc al final del estudio en vida. Los cerebros se seccionaron en rodajas muy delgadas para que las proteínas específicas de las células pudieran marcarse con el uso de anticuerpos. A las 2 semanas, algunas células expresaron la proteína informadora, y la expresión aumentó progresivamente a las 8 semanas e incluso más a las 20 semanas después de la entrega del gen. Estos datos mostraron una expresión estable y a largo plazo del ARNhc.

Una preocupación en la terapia génica es si la expresión a largo plazo del gen puede inducir consecuencias desfavorables para las células del cerebro. Usando anticuerpos para marcar las proteínas neuronales, los autores encontraron que, si bien 2 semanas después de la inyección había una pérdida clara de neuronas en el lugar de la inyección, en los puntos de tiempo posteriores, esta pérdida ya no era aparente. Los autores del estudio propusieron que esta recuperación podría deberse al nacimiento de nuevas neuronas y / o al proceso de brote neuronal donde las neuronas generan ramas adicionales que hacen contacto con las neuronas vecinas.

Un factor limitante importante para la terapia génica es la respuesta inmune del huésped, que se activa cuando el cuerpo ve al nuevo vector como un invasor extraño. En este estudio, los investigadores observaron señales inflamatorias en el cerebro. La microglía y los astrocitos son dos tipos de células en el cerebro que se activan tras una lesión e inflamación. La actividad astrocítica y microglial aumentó poco después de la inyección solo en animales donde se inyectó el ARNhc contra la ataxina-3 mutante. A las 8 y 20 semanas, sus niveles volvieron a los niveles observados en los ratones no inyectados. Un tipo especial de proteínas inflamatorias, conocidas como citocinas, se elevaron después de la inyección, pero también volvieron a los niveles de control a las 20 semanas. Juntos, los resultados mostraron que incluso cuando la inflamación se desencadenó al principio del curso de la terapia, se disipó,

Este fue un estudio cuidadosamente realizado para evaluar el perfil de seguridad de un candidato a terapia génica para SCA3. En su estudio anterior de prueba de concepto, los autores demostraron que la reducción de los niveles de Ataxin-3 mutante mejoró varias características anormales en modelos de ratón SCA3. Este estudio actual muestra que el uso de este agente terapéutico en ratones de tipo salvaje es seguro hasta 20 semanas después de la administración de la terapia génica. Si bien este estudio utilizó una inyección localizada del agente de terapia génica solo en el cuerpo estriado, varias regiones del cerebro se ven afectadas en SCA3. Es necesario un estudio adicional que utilice una ruta de administración que se dirija a múltiples regiones del cerebro para evaluar el perfil de seguridad.

Son necesarios estudios futuros para caracterizar el perfil de expresión y la seguridad en primates no humanos. La vía de administración sería similar a la de los pacientes humanos, lo que permitiría que los resultados fueran más traducibles para ensayos clínicos. Este es un momento verdaderamente emocionante para la terapia génica, pero también es importante mantener la seguridad de los pacientes como una prioridad absoluta.

Términos clave

Gen: una unidad de la herencia que contiene nuestro ADN, el código que controla el desarrollo y la función de nuestro cuerpo.

Monogénico: trastorno o enfermedad involucrada o controlada por un solo gen.

Vector: un modo de transporte para llevar material genético extraño a otra célula.

ARN: ácido nucleico que transporta instrucciones del ADN para producir proteínas.

ARN en horquilla corta: un tipo de ARN plegado en una estructura en horquilla que puede apuntar a los genes y silenciarlos.

Declaración de conflicto de intereses

El autor y el editor declaran no tener ningún conflicto de intereses.

Cita del artículo revisado

Nóbrega, C, et al. RNA interference therapy for Machado-Joseph Disease: Long-term safety profile of lentiviral vectors encoding short hairpin RNAs targeting mutant Ataxin-3. Human Gene Therapy, 2019. 30:7 https://doi.org/10.1089/hum.2018.157

Evaluating the long-term safety of lentiviral gene therapy in SCA3 mice

Written by Dr. Ambika Tewari Edited by Dr. Hayley McLoughlin

Lentiviral expression of an shRNA against ataxin-3 was well-tolerated and produced no measurable adverse effects in wild-type mice.

Evaluating the safety profile is a necessary and crucial step in qualifying a therapy for use in patients. Gene therapy is an experimental technique that has demonstrated tremendous progress in the treatment or reversal of a disease, specifically monogenic disorders. Carefully investigating the safety and tolerance of gene therapy is important to gauge its suitability for clinical trials. Gene therapy tools can be used in different ways to achieve the same therapeutic effect: the faulty gene can be replaced with a healthy copy, the mutated gene can be repaired, or the mutant copy of the gene can be silenced. You can learn more about gene therapy in this pat SCAsource Snapshot.

Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) causes progressive loss of neurons in the spinal cord, and several regions of the brain. This includes the cerebellum, brainstem, striatum and substantia nigra. These neurons have crucial functions. Without these neurons, patients experience motor incoordination, loss of balance, and in severe cases, premature death. While great progress continues to be made in understanding how a mutation in a single gene, Ataxin-3, causes the symptoms of SCA3, there is still no treatment to stop the disease progression. As a monogenic disorder, SCA3, like other Spinocerebellar ataxias (SCA), is a promising candidate for gene therapy. While there are no approved gene therapies for SCA yet, there any several research labs and companies working towards achieving this goal.

An artist's drawing of scientists standing infront of a giant piece of DNA and drugs
This is truly an exciting time for gene therapy, but it is also important to keep the safety of patients a top priority. Photo used under license by Visual Generation/Shutterstock.com.

The researchers in this study have been working on gene therapy for SCA3 since 2008. They have researched how gene therapy could offer protection against further decline, in several cell and mouse models of SCA3. They used an approach where they decreased the levels of the mutant Ataxin-3 gene while leaving the normal Ataxin-3 gene intact. This is known as allele-specific targeting. They demonstrated that using this technique, they could significantly reduce the behavioral and neuropathological changes that occur in SCA3 mice. Mice treated with the gene therapy showed improvements in their balance and motor coordination. 

Gene therapy in its most basic form involves two components, the gene that will replace or remove the diseased gene and a vector that will transport this new gene to its site of action. The most commonly used vectors today are adeno-associated virus (AAVs) followed by retrovirus. These viruses have been specifically engineered to deliver their passenger to the specified location. While both vectors have been through several years of preclinical and clinical testing for numerous gene therapy candidates, there are questions that remain regarding their safety. (1) Does the gene therapy product continue to be expressed in the targeted area long-term; (2) If there is long-term expression does it cause any adverse measurable effects to the targeted area; (3) Does the long-term expression affect the normal functioning of the targeted cells/organ.

Continue reading “Evaluating the long-term safety of lentiviral gene therapy in SCA3 mice”

Regulating ataxin-1 expression as a therapeutic avenue for SCA1

Written by Dr. Hannah Shorrock   Edited by Dr. Hayley McLoughlin

Nitschke and colleagues identify a microRNA that regulates ataxin-1 levels and rescues motor deficits in a mouse model of SCA1

What if you could use systems already in place in the cell to regulate levels of toxic proteins in disease? This is the approach that Nitschke and colleagues took to identify the cellular pathways that regulate ataxin-1 levels. Through this strategy, the group found a microRNA, a small single-stranded RNA, called miR760, that regulates levels of ataxin-1 by directly binding to its mRNA and inhibiting expression. By increasing levels of miR760 in a mouse model of SCA1, ataxin-1 protein levels decreased and motor function improved. This approach has the potential to identify possible therapies for SCA1. It may also help identify disease-causing mutations in ataxia patients with unknown genetic causes.

Spinocerebellar Ataxia type 1 (SCA1) is an autosomal dominant disease characterized by a loss of coordination and balance. SCA1 is caused by a CAG repeat expansion in the ATXN1 gene. This results in the ataxin-1 protein containing an expanded polyglutamine tract. With the expanded polyglutamine tract, ataxin-1 is toxic to cells in the brain and leads to dysfunction and death of neurons in the cerebellum and brainstem.

As with all protein-coding genes, surrounding the protein coding region of ATXN1 gene are the 5’ (before the coding sequence) and 3’ (after the coding sequence) untranslated regions (UTRs). These regions are not translated into the final ataxin-1 protein product but are important for the regulation of this process. Important regulation factors called enhancers and repressors of translation located in 5’ and 3’ UTRs. ATXN1 has a long 5’ UTR. Genes that require fine regulation, such as growth factors, are often found to have long 5’ UTRs: the longer a 5’ UTR, the more opportunity for regulation of gene expression. The group, therefore, tested the hypothesis that the 5’ UTR is involved in regulating the expression of ataxin-1.

In their initial studies, Nitschke and colleagues identified that the ATXN1 5’UTR is capable of reducing both protein and RNA levels when placed in front of (5’ to) a reporter coding sequence. One common mechanism through which this regulation of gene expression could be occurring is the binding of microRNAs, or miRNAs, to the ATXN1 5’UTR. miRNAs are short single-stranded RNAs that form base pairs with a specific sequence to which the miRNA has a complementary sequence; this leads to regulation of expression of the mRNA to which the miRNA is bound.

3d illustration of single-strand ribonucleic acid
Artist drawing of single-stranded RNA. Photo used under license by nobeastsofierce/Shutterstock.com.

Using an online microRNA target prediction database called miRDB, the group identified two microRNAs that could be responsible for these changes in gene expression through binding to the ATXN1 5’ UTR. By increasing the expression of one of these microRNAs, called miR760, ataxin-1 protein levels were reduced in cell culture. Conversely, using a miR760 inhibitor so that the miRNA could not perform its normal functions led to increased levels of ataxin-1. Together this shows that miR760 negatively regulates ataxin-1 expression.

Continue reading “Regulating ataxin-1 expression as a therapeutic avenue for SCA1”

Discovery of a new molecular pathway in spinocerebellar ataxia 17

Written by Dr. Sriram Jayabal Edited by Dr. Ray Truant

A potential new pathway for SCA17: gene therapy that in mice restores a critical protein deficit protects brain cells from death in SCA17.

Neurodegenerative ataxias are a group of brain disorders that progressively affect one’s ability to make fine coordinated muscular movements. This makes is difficulty for people with ataxia to walk. Spinocerebellar ataxia type 17 (SCA17) is one such late-onset neurological disease which typically manifests at mid-life. The life expectancy after symptoms first appear is approximately 18-20 years. Besides ataxia, SCA17 can cause a number of other symptoms ranging from dementia (loss of memory), psychiatric disorders, dystonia (uncontrollable contraction of muscles), chorea (unpredictable muscle movements), spasticity (tightened muscles), and epilepsy.

Brain imaging and post-mortem studies have identified that the cerebellum (often referred to as the little brain) is one of the primary brain regions that is affected. That being said, other brain regions such as the cerebrum (cortex or the big brain) and brainstem (distal part of the brain found after the cerebellum) could undergo degeneration. Further, the genetic mutation that leads to SCA17, is a CAG-repeat expansion mutation, similar to several other forms of ataxias. In most other ataxias, where the function of the mutated protein is unknown. However in SCA17, the function of the mutated protein, TATA-box binding protein, is very well understood. Despite this unique advantage, we are yet to completely understand how the mutant gene leads to SCA17. This is why current treatment strategies often focus on treating the symptoms, but not the underlying cause.

person holding laboratory flask
Photo by Chokniti Khongchum on Pexels.com

SCA17 mutation leads to Purkinje cell death

Researchers from China have shed more light on how the mutant gene causes SCA17. TATA-box binding protein is a transcription initiation factor is a protein that turns on the production of RNA from genes. It is widely found across the brain including the cerebellum. TATA-box binding protein controls the amount of protein manufactured from several genes. This raised a very important question: pertinent not only to SCA17 but also more generally to several SCAs – why is that the cerebellar neurons, especially the most sensitive neuron, the Purkinje cells die?

Continue reading “Discovery of a new molecular pathway in spinocerebellar ataxia 17”

Snapshot: The next-generation of CRISPR is prime editing – what you need to know

The CRISPR gene-editing toolbox expanded with the addition of prime editing. Prime editing has astounding potential for both basic biology research and for treating genetic diseases by theoretically correcting ~89% of known disease-causing mutations.

What is prime editing?

Prime editing is coined as a “search-and-replace” editing technique that builds on the “search-and-cut” CRISPR technology. Like CRISPR, prime editing utilizes the Cas9 enzyme targeted to a specific location in the genome by a guide RNA (gRNA). With a few ingenious modifications, including an enzyme called a reverse transcriptase (RT) fused to Cas9, prime editors can be targeted to nearly anywhere in the genome where the RT writes in new DNA letters provided by a template on the gRNA.

graphic drawing of red handled scissors
New gene-editing techniques offer more opportunities for therapy development. Each new discovery makes the techniques more and more accurate. Image courtesy of yourgenome.

 How is prime editing different from CRISPR?

Scientists are excited about prime editing because it has several advantages and overcomes many of the limitations of previous CRISPR systems. CRISPR Cas9, an endonuclease, cuts—like scissors—both DNA strands to inactivate a gene or to insert a new sequence of donor DNA. Unlike CRISPR edits, the prime editing Cas9, a nickase, cuts a single DNA strand and does not rely on the cell’s error-prone repair machinery, thereby minimizing any resulting deleterious scars left on the DNA. It has a broader range of targets because it is not limited by the location of short DNA sequences required for Cas9 binding to DNA. The versatility and flexibility of the system allows for more control to inactivate genes as well as to insert, remove, and change DNA letters, and, combine different edits simultaneously—analogous to a typewriter. Importantly, the edits are precise with relatively infrequent unwanted edits. Initial indications showed fewer off-target edits in the genome, possibly because more steps are required for a successful edit to occur. In some cases, it may be more efficient than CRISPR, depending on the targeted cell type, such as in a non-dividing cell like a neuron in the brain. However, with all these advantages, CRISPR still remains the tool of choice for making large DNA deletions and insertions because the prime editing system is limited by the RT and template RNA length.

How could prime editing help ataxia patients?

Prime editing offers enormous possibility for correcting heritable ataxia mutations accurately and safely. In dominantly inherited SCAs, like SCA1 or SCA2, prime editing could shorten the pathogenic repeat expansion allele to the normal length, or inactivate the pathogenic allele without creating unwanted, deleterious mutations. It also provides researchers with a powerful tool to study disease-causing genes in cells and animal models in new ways to advance our knowledge about the underlying mechanisms in ataxia.

What barriers are there to using prime editing as a treatment?

Prime editing will require rigorous testing in cells and animals before moving into humans in a clinical trial. Optimizing delivery and efficiency in target cells and tissues, and minimizing side-effects will be the key barriers to overcome.

To read the original Nature article describing prime editing, it can be found from the Liu lab here.

If you would like to learn more about Prime Editing, take a look at these news stories by The Broad Institute and Singularity Hub.

Snapshot written by Bryan Simpson and edited by Dr. Hayley McLoughlin.