Snapshot: O que é terapia genética?

Terapia genética significa utilizar ácidos nucleicos para tratar uma desordem genética. Esses ácidos nucleicos podem ser desenhados em uma variedade de formas para alcançar um mesmo propósito terapêutico. Ferramentas de terapia genética podem ser utilizadas para corrigir um gene mutante através de uma das três formas:

  1. Expressão de uma cópia saudável do gene
  2. Silenciamento ou ativação do transcrito de um gene mutante
  3. Utilização de ferramentas de edição genética para reparar ou desligar o gene mutado.
computer desk laptop stethoscope
Foto de um estetoscópio, por Negative Space no Pexels.com

Como a terapia genética é utilizada?

Doenças monogênicas, como algumas ataxias espinocerebelares (SCAs) são alvos excelentes para abordagens de terapia genética. Terapias genéticas são atualmente utilizadas em pesquisas em ataxia para estudar os mecanismos das doenças e para a aplicação terapêutica em ensaios pré-clínicos.

Terapia genética. 1. Um gene saudável, RNA de interferência ou ferramentas de edição gênica são “empacotados” em vírus adeno-associados (mas também podem ser entregues à célula somente como molécula de DNA ou em nanopartícula). 2. As partículas virais são injetadas no tecido de interesse. 3. O virus entra nas células e libera o material genético. A célula então se tornará saudável por uma das seguintes vias: 1) expressão do gene normal; 2) repressão do RNA mutante, ou 3) correção do gene mutante.
Visão geral da terapia genética, desenvolvida pela Stephanie Coffin com uso do Biorender.

Uma abordagem de terapia genética para resgatar o fenótipo em SCA1 envolve a expressão do gene saudável, ataxina-1-like, que compete com a proteína ATXN1 mutante pela formação de complexos proteicos. Esse trabalho, conduzido por Keiser e colaboradores em 2016, mostrou o resgate fenotípico em um modelo de camundongo de SCA1.

Existem duas tecnologias comuns utilizadas para o silenciamento ou inativação de genes relacionados a doenças: RNA de interferência (RNAi) ou oligonucleotídeos antisenso (ASOs). As estratégias de RNAi utilizam pequenas moléculas de RNA para silenciar a expressão de transcritos-alvo de RNA mutante, enquanto os ASOs são moléculas de DNA usadas para silenciar ou corrigir transcritos de RNA mutante. Ambas as abordagens terapêuticas estão sendo testadas em SCAs. Por exemplo, Carmo e colaboradores mostraram em 2013  que a utilização de RNAi tendo como alvo o gene que causa SCA3, ATXN3, reduziu longitudinalmente os níveis de ATXN3 mutante. Veja o SCAsource snapshot sobre ASOs para maiores informações sobre seu uso em SCAs.

A ferramenta mais comum de edição genética é o sistema CRISPR/Cas9, que utiliza um guia de RNA para direcionar a enzima nuclease Cas9 para a região do genoma a ser editada. Desse modo, o gene mutante pode ser removido ou corrigido. Ainda é cedo para essa tecnologia ser considerada como potencial terapêutico, devido aos desafios enfrentados para a inserção do sistema na célula e os riscos de edição de genes não-alvos.

Como a terapia genética é entregue à célula?

Um dos aspectos mais difíceis da terapia genética é a inserção das diversas moléculas necessárias nas células de interesse. Um método comum é através da utilização de vírus. Nesse método, o ácido nucleico é transferido para as células doentes através de um vetor, que é um vírus que foi modificado para a retirada dos componentes virais. Os vetores virais mais comuns atualmente utilizados na terapia genética são os vírus adeno-associados (AAVs). Outros métodos de inserção incluem vetores não-virais, como moléculas de DNA e nanopartículas.

Qual a durabilidade da terapia genética?

A inserção dos produtos da terapia genética por meios virais promove uma expressão longitudinal do ácido nucleico, enquanto a inserção direta da molécula de DNA ou nanopartículas resulta em uma expressão transiente do ácido nucleico e, portanto, tipicamente requer um tratamento contínuo.

Gostaria de aprender mais sobre terapia genética? Dê uma olhada no conteúdo (em inglês) dos sites do National Institutes of Health e KidsHealth.

Snapshot escrito por Stephanie Coffin e editado por Dr.Hayley McLoughlin. Inicialmente publicado em 23 de agosto de 2019. Traduzido para o português por Priscila P. Sena.

¿Qué es el nistagmus?

El nistagmus, también conocido como ataxia ocular, es un término que se refiere al movimiento incontrolable del ojo, generalmente un ciclo repetitivo de movimiento lento en una dirección específica seguido de un ajuste rápido de regreso al centro. La raíz de este movimiento reside en un reflejo normal que usamos todos los días: el reflejo vestibulo-ocular. Este reflejo controla cómo nuestro sentido del equilibrio y el movimiento de la cabeza (nuestro sentido ‘vestibular’) dirige el movimiento de nuestros ojos (el componente ‘ocular’ se refiere a los músculos del ojo).

Por ejemplo, si miramos algo como la barra espaciadora de nuestro teclado y movemos la cabeza lentamente hacia adelante y hacia atrás, nuestros ojos generalmente pueden permanecer fijos en la barra espaciadora sin mucho esfuerzo consciente. Esto ocurre debido a la comunicación constante entre nuestro oído interno y los músculos de nuestros ojos mientras nuestra cabeza se mueve en el espacio.

Para ser un poco más técnico sobre cómo funciona esto, tenemos órganos sensoriales especiales llamados » canales semicirculares » en el oído interno que sirven como un giroscopio biológico. A medida que gira la cabeza en una dirección determinada, el fluido en estos canales cambia en relación con su movimiento. El desplazamiento de este fluido activa neuronas especializadas que a su vez activan otras neuronas para obtener la información de cómo se está girando desde el oído, al cerebelo, a los músculos que controlan el ojo. Sin embargo, hay circunstancias en las que esta línea de comunicación puede verse abrumada o interrumpida. Esta interrupción hace que nuestros ojos se muevan a pesar de que nuestras cabezas están quietas. Cuando esto sucede, tenemos nistagmus.

Por ejemplo, aquí hay un video de alguien que experimenta el reflejo vestíbulo-ocular mientras gira en una silla y nistagmus después de girar en una silla . En este caso, el nistagmus ocurre cuando la persona deja de dar vueltas en la silla porque el líquido en el oído interno continúa moviéndose por un corto tiempo a pesar de que la cabeza se ha detenido.

women is looking into the camera, her eyes show shee is looking to the side.
El nistagmus es un término que se refiere al movimiento incontrolable del ojo. Foto utilizada bajo licencia por Wanchana Olena Yakobchuk/Shutterstock.com.

La ataxia, la pérdida del movimiento coordinado, es causada por la degeneración del cerebelo. Una de las funciones principales del cerebelo es como centro de integración de cómo usamos la información sensorial entrante (tacto, vista, equilibrio, etc.) para dirigir cómo nos movemos en el espacio. Por lo tanto, vemos que a medida que la ataxia empeora, los movimientos voluntarios complejos como caminar se vuelven más difíciles de controlar. Esto también puede alterar el funcionamiento de los reflejos que utilizan el equilibrio y el movimiento de la cabeza, como el reflejo vestíbulo-ocular. A medida que se mueve la cabeza, la información sobre cómo se mueve la cabeza va inicialmente a un área específica del cerebelo que luego le dice a los músculos oculares cómo moverse.

Cuando las células cerebelosas de Purkinje de esa zona dejan de funcionar correctamente, este canal de comunicación se vuelve hiperactivo. Los músculos del ojo comienzan a moverse esporádicamente como si la cabeza se estuviera moviendo o girando, aunque estuviera quieta. Este es un síntoma importante a tratar en pacientes con ataxia. El nistagmus altera la vista y está relacionado con síntomas secundarios como mareos y náuseas. Esta combinación de síntomas obstaculiza gravemente la independencia de una persona y reduce su calidad de vida.

Si desea obtener más información sobre el nistagmus, consulte estos recursos de Johns Hopkins y la Academia Estadounidense de Oftalmología .

Escrito por Carrie Sheeler y editada por el Dr. Siddharth Nath. Publicado inicialmente en el 10 de diciembre de 2021. Traducción al español fueron hechas por FEDAES

Snapshot: What is the Pole Test?

The pole test is a common and straightforward test to assess motor coordination in mice. While ataxia might be easy to see in patients, it is not always as apparent in ataxia mouse models. Therefore, this fast and simple test is important for researchers to measure disease severity. It is also important to test the effect of different treatment strategies.

Small experimental mouse is on the laboratory researcher's hand with blue gloves
 Photo used under license by unoL/Shutterstock.com.

How is the pole test performed?

At the beginning of the test, the mouse is placed facing upward on the top of a long pole. The researchers then measure the time the mouse takes to turn around and climb down to the bottom of the pole. A healthy mouse typically takes 10-20 seconds to perform the task. If the mouse struggles and takes a long time to get to the bottom, it suggests that the mouse has motor coordination deficits.

Researchers commonly use the pole test because it’s a quick way to assess coordination in mice, even before the mice show obvious ataxia symptoms. The pole test takes about 5 minutes per mouse. It is thereby much faster than other motor coordination tests, such as the rotarod test, typically performed over multiple days. Another advantage is that the pole test can be repeated on the same mice multiple times. This allows for tracking how a mouse’s motor coordination changes over time.

0 seconds - mouse is at top of a pole facing upward. 5 seconds - mouse climbs to the top of the pole to turn around, so it can face down towards the ground. 10 seconds - mouse has climbed down the pole
Cartoon of mouse performing the pole test. Time is shown in seconds. Image courtesy of Eder Xhako.

How is the pole test used in literature?

One example of the pole test being used in the literature is a study by Nitschke and colleagues. In this study, the researchers identified a small regulatory RNA, miR760, that regulates the levels of ATXN1. ATXN1 is the gene that causes Spinocerebellar Ataxia Type 1 (SCA1). The group showed that injections of miR760 in the brain decreases ATXN1 protein levels in a SCA1 mouse model. The researchers then used the pole test to measure how the treatment with miR760 would affect the ataxia phenotype in the SCA1 model. They found that one month after the treatment the mice displayed improved motor coordination compared to control mice.  

If you would like to learn more about the Pole Test, take a look at this resource by Melior Discovery. You can learn more about other motor coordination tests in our past Snapshots on the Rotarod Test.

Snapshot written by Eder Xhako and edited by Dr. Larissa Nitschke.

Snapshot: What is the International Cooperative Ataxia Rating Scale?

The International Cooperative Ataxia Rating Scale (ICARS) is an assessment of the degree of impairment in patients with cerebellar ataxia. It was developed in 1997 by the Committee of the World Federation of Neurology. The goal of ICARS is to provide a standardized clinical rating score to measure the efficacy of potential treatments. The scale was intended for patients with cerebellar ataxia. But ICARS has also been validated for patients with focal cerebellar lesions, spinocerebellar, and Friedrich’s ataxia.

How Does it Work?

The ICARS is a semi-quantitative examination that translates the symptomatology of cerebellar ataxia into a scoring system out of 100. The assessment is designed to be completed within 30 minutes, and higher scores indicate a higher level of disease impairment. The assessment consists of 19 items and four subscales of postural and gait disturbances, limb movement disturbances, speech disorders, and oculomotor disorders. Detailed descriptions of the scoring metrics are also provided to reduce scoring variability between the examiners.

Advantages and Drawbacks

Since its development, multiple studies have validated the ICARS. It has also been widely used in clinical assessment for ataxia rating of different diseases. One such study accessed 14 instruments of ataxia assessment and identified the ICARS to be highly reproducible and internally consistent.

However, the scale also does not account for some ataxia symptoms, such as hypotonia (muscle weakness), that are difficult to access clinically. Some subscales also have a considerable ceiling effect, where many patients reach the maximum score for a category. This means symptoms are not being accessed past a certain severity.

Doctor writing down patient notes on a clipboard using a checklist while sitting at a desk.
The ICARS is a semi-quantitative examination that translates the symptomatology of cerebellar ataxia into a scoring system out of 100. Photo used under license by eggeegg/Shutterstock.com.

Other Ataxia Rating Scales

The Scale for the Assessment and Rating of Ataxia (SARA) is another semi-quantitative assessment of impairment levels. It consists of only eight items, making it easier to perform for frequent assessments. However, the simplification of the scale excludes some important symptomatology, including oculomotor impairment.

A pilot study has also been conducted for the development of SARAhome, a video-based variation of SARA that can be conducted independently at home, showing promise for the digitization of ataxia assessment.

Another assessment scale that is even more toned-down is the Brief Ataxia Rating Scale (BARS). The scale consists of five items that assess gait, speech, eye movement, and limb mobility, and the estimated assessment time is only five minutes.

All the assessments described above have been validated and each has its own benefits and drawbacks. However, none of them provides the minimal important difference, which is an important clinical measurement used to determine the effectiveness of potential treatment. Therefore, we are still in need of developing better tools for measuring disease impairment in ataxia patients.

If you would like to learn more about ICARS, take a look at this resource by Physiopedia.

Snapshot written by Christina (Yi) Peng and edited by Dr. Hayley McLoughlin.

Snapshot: What is N-acetylcysteine?

What is N-acetylcysteine used for?

Cysteine is an amino acid that is used as a building block in our bodies to make proteins. We consume cysteine in our diets through protein-rich foods, like beef or lentils. N-acetylcysteine is a chemical derivative of cysteine. This means which means that N-acetylcysteine contains one change in its chemical structure that distinguishes it from cysteine. N-acetylcysteine is often taken as a supplement. It is also used clinically by doctors to treat patients experiencing acetaminophen ( also known as Tylenol or paracetamol) overdose and some respiratory conditions such as bronchitis.

Companies that sell N-acetylcysteine as a supplement claim that it can prevent or treat many health ailments, such as cancer, liver disease, diabetes, high cholesterol, and psychiatric disorders like depression. Some clinical trials have been conducted to test if N-acetylcysteine can truly help with these illnesses, but the results have been mixed and inconclusive.

Some of this variability could come from the way N-acetylcysteine was given to the participants (orally, nasally, or intravenously) and the dosage amount. While N-acetylcysteine is safe to consume, it’s worth noting that some participants in these trials have had more adverse effects than placebo groups. They weren’t serious, but usually consisted of nausea and headache.

White capsule pills spilling out of a percription bottle.
N-acetylcysteine is being tested to treat neurodegenerative diseases, but the clinical trial results have been mixed and inconclusive. Image used from Pxfule.

How does our body utilize N-acetylcysteine?

The main way N-acetylcysteine is thought to provide a therapeutic effect in the body is by acting as an antioxidant. Antioxidants are substances that prevent or slow the damage that can be caused to cells by free radicals, which are also known as reactive oxygen species. Free radicals are molecules that have an unpaired electron. This causes free radicals to be unstable because they want to “steal” an electron from a nearby molecule so that their unpaired electron will have a partner. If left unchecked, free radicals could damage cell components like lipids and nucleic acids (like DNA) through this process.

Fortunately, our body has antioxidant defenses to prevent this damage as they can neutralize free radicals. One of the main antioxidants our body uses is glutathione. To produce glutathione, a cysteine molecule is required. N-acetylcysteine can provide that cysteine to increase the levels of glutathione. This may be helpful for diseases where oxidative stress is involved in disease pathology, like in neurodegenerative or heart diseases. Oxidative stress occurs when there is an imbalance between free radicals and antioxidants, leading to an accumulation of free radicals.

Has N-acetylcysteine ever been tested as a treatment for Spinocerebellar ataxia (SCA)?

There have not been any clinical trials conducted to test the effect of N-acetylcysteine on any of the spinocerebellar ataxias. The only experimental data that exists is from a study in 2003 that used a cell model for SCA1, where researchers found that N-acetylcysteine had a positive effect on cell traits associated with SCA1. However, there have been some clinical trials in other neurodegenerative diseases. Oxidative stress in the brain is commonly seen in many neurodegenerative diseases, including many types of spinocerebellar ataxia and Parkinson’s disease.

Several clinical trials have been done in Parkinson’s disease to determine if N-acetylcysteine can help improve symptoms, but there have been some conflicting results that may be due to the way N-acetylcysteine was administered. When participants took N-acetylcysteine only orally, there was no effect on symptoms and brain scans did not show increased antioxidant levels. However, in a trial where oral was combined with intravenous administration, some positive effects on symptoms and biomarkers were found. You can learn more about this study at this link. More information about this study can be found here.

For now, we cannot make conclusions that N-acetylcysteine would have the same effect for all neurodegenerative diseases, but it does have potential that should be explored by researchers in spinocerebellar ataxias.

If you would like to learn more about N-acetylcysteine, take a look at this resource by the Memorial Sloan Kettering Cancer Center.

Snapshot written by Nola Begeja and edited by Dr. Gulin Oz.