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The identification of genetic defects that underlie inherited retinal diseases (IRDs) paves the way for the development of therapeutic strategies. Nonsense mutations caused approximately 12% of all IRD cases, resulting in a premature termination codon (PTC). Therefore, an approach that targets nonsense mutations could be a promising pharmacogenetic strategy for the treatment of IRDs. Small molecules (translational read-through inducing drugs; TRIDs) have the potential to mediate the read-through of nonsense mutations by inducing expression of the full-length protein. We provide novel data on the read-through efficacy of Ataluren on a nonsense mutation in the Usher syndrome gene USH2A that causes deaf-blindness in humans. We demonstrate Ataluren´s efficacy in both transiently USH2AG3142*-transfected HEK293T cells and patient-derived fibroblasts by restoring USH2A protein expression. Furthermore, we observed enhanced ciliogenesis in patient-derived fibroblasts after treatment with TRIDs, thereby restoring a phenotype that is similar to that found in healthy donors. In light of recent findings, we validated Ataluren´s efficacy to induce read-through on a nonsense mutation in USH2A-related IRD. In line with published data, our findings support the use of patient-derived fibroblasts as a platform for the validation of preclinical therapies. The excellent biocompatibility combined with sustained read-through efficacy makes Ataluren an ideal TRID for treating nonsense mutations based IRDs.

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Abstract. Choroideremia (CHM) is an x-linked recessive chorioretinal dystrophy, with 30% caused by nonsense mutations in the CHM gene resulting in an in-frame

Quelle: Nonsense-mediated mRNA decay efficiency varies in choroideremia providing a target to boost small molecule therapeutics

Mariya Moosajee Klinischen Studien für Behandlung von Nonsense-Mutationen bei USH2a

Untenstehende Fragen wurden bei der Konsultation am 18.Oktober 2018
detailliert und sehr aufschlussreich beantwortet.

(1) patient registries
Does England have its own patient registry for retinal patients,
such as the American >My Retina Tracker< ?
The German self-help association Pro Retina has implemented one
for its members.
This registry will facilitate access to clinical trials for patients and help researchers recruit patients.
I have already suggested that <My Retina Tracker> and the German Registry should discuss whether it would not be useful for researchers if such a Registry included mutations such as missense or nonsense. Therefore, it would be easier to reach suitable patients with nonsensical mutations, especially in one of their research areas. How do you see this?

(2) changes during pregnancy and puberty
The affected siblings from Germany report that during puberty both had improved structural hair growth.
During pregnancy, Mrs. XXX once again experienced a noticeable positive change.
To what extent are factors known that could lead to these noticeable
changes during pregnancy?
It is known that chemical processes are increased during puberty or pregnancy.
Are you aware of any results in these cases which, for example, explain the epigenetic factors in retinal diseases?

I have the following further question on this subject.
Is it conceivable that during this phase of physical change,
where the growth behaviour of the hair changes,
there is also a slowed rate of decay of the photoreceptors ?
In the case of known patients, among others in the UK,
it would be desirable to observe this point in time more closely with regard to this thesis.
Are you aware of any changes in other retinal diseases during puberty and pregnancy?

(3) progression forms
I have compiled an overview table of all publications on HJMD available to me which contained information on visual acuity.
 I have attached this table as an attachment.
(The values consist of 2 points: 1x visual acuity at diagnosis and 1x visual acuity at last examination.In some cases there are many years in between.Unfortunately there are not 2 points for all patients.
All values have been converted to decimal values.)
Here it can be deduced that in the vast majority of cases there is a slow deterioration in vision.
Nevertheless, there are some conspicuous features.
It seems that those with good vision at a younger age tend to retain it even at an advanced age, in correlation with the other processes.

More data will certainly be needed to provide a more accurate prognosis of the progression. In the meantime I learned that a representation in decimal values is not correct and above all should not be represented linearly graphically. I will next convert the results of the patients presented to you into LogMar values and create a new overview.

In this context, the following question arises: XXX and XXX, the siblings who have identical mutations in the CDH3 gene, have so different visual acuity values. XXX is much older, but also has a much better visual acuity.
What are the underlying factors why there is such a variety of progressions, even with identical mutations?

(4) EEM-Syndrom
Do you have an explanation why the EEM syndrome,
which is based on the same genetic defect, is accompanied by pronounced ectodermal malformations?
After studying the publications on the syndrome,I put forward the thesis that HJMD and EEM syndrome are the same disease, but HJMD is a milder form.
Also with the HJMD there is the occurrence of ectodermal changes.

(5) antibodies
Do you know if he already gives antibodies for the detection of the CDH3 gene? These antibodies are, as far as I understand it, used for testing e.g. TRIDs to detect the missing expression.

(6) hair phenotype
The hair phenotype in HJMD patients is an essential feature,
the hair growth cycle can normally be detected optically over a few weeks,
so that the HJMD would be ideal for testing the efficacy in nonsense mutations, or ?
The proof of photoreceptors seems to me to be more tedious here.
Do you have knowledge of investigations in the field of hereditary hair growth disorders based on this genetic basis. 

(7) fibroblasts
With Mrs. Doctor XXX from Wolfrum Lab,Mainz I have already talked personally about her work on USH2 several times.
Meanwhile a Usher pig was drilled there.
Is there actually an international exchange among you as researching doctors ?
In patients with various genetic mutations, do you usually also take skin samples known as fibroblasts for the purpose of demonstrating efficacy ?

(8) Aniridia
PTC124 and several other designers aminoglycoside are meanwhile very familiar to me. I'm following the clinical study >Study of Ataluren in Patients With Aniridia (STAR)< with excitement.
Are you already aware of the first interim results, about the efficacy ? Is it correct that in this study PTC124 is taken exclusively orally?
Orally administered, the active substance should probably fail at the blood retina barrier, or ?
I therefore do not necessarily understand the point of this study if the active ingredient is administered orally rather than not by drop.
At clinical trials the administration of the study is clearly described orally.
https://clinicaltrials.gov/ct2/show/NCT02647359?cond=aniridia&rank=1thBut other sources report that the active substances are administered with drops. http://ffb.ca/scientists-are-developing-an-eye-drop-to-treat-inherited-blinding-eye-diseases/
This is confusing.

(8) blood retina barrier
Various publications report on possible parmakological therapy approaches in connection with liposomes. It is intended to facilitate administration into the retina.
How do you assess the state of affairs here?

(9) eye drops
The administration of eye drops, which would certainly have to be taken daily, would be a great option here.
Is it possible that active substances diffuse through the dermis and choroid?

(10) Ataluren
There are 3 stop codons.
Ataluren does not react immediately to these three codons.
You get the best results with the code UGA.
Then follow the UAG and the UAA. Another important effect that affects effectiveness is seen in the first letter of the following codon.
If the following codon starts with the letter C,
this is the best prerequisite for the effectiveness of atalures.
Have I understood this so far?
Do you already have experience values such as such deviations show up in their effectiveness? Is it the same for other aminoglycosides?
I think my son XXX has a UGA nonsense, is that right?
c.1086G>A /  pTrp362*

(11) aminoglycosides
Which active substances do you evaluate with regard to their suitability for the most promising and do you already have practical knowledge about ELX-02 ?

The effect of PTC124 on choroideremia fibroblasts and iPSCderived RPE raises considerations for therapy

Ein Unternehmen aus Israel namens Eloxx Pharmaceuticals, eine 5 Millionen Dollar Finanzspritze aus dem Kapitalmarkt, um die Forschung von Therapieoptionen basierend auf Nonsense Mutationen zu intensivieren.

externer Link: Wallstreet Online

Eloxx Pharmaceuticals ist ein Biotechnologieunternehmen mit Schwerpunkt auf der Entdeckung, Entwicklung und Vermarktung von Verbindungen für die Behandlung genetischer Krankheiten aufgrund von Nonsensmutationen.

externer Link: Unternehmenswebseite

Durch die Fortschritte in der Gentherapie soll es möglich sein, spezielle Therapien für ganz individuelle Gendefekte zu entwickeln. 12% aller vererbten genetischen Störungen entstehen durch sogenannte Nonsense-Mutationen. Bei einigen Erkrankungen, wie zum Beispiel der Chorioideremie (Netzhauterkrankung), geht man sogar von über 30% Prozent aus.

Durch diese Mutationen wird ein vorzeitiger Abbruch der Proteinherstellung verursacht.  Das heißt, die normale vollständige Proteinherstellung wird unterbunden.

Eine Therapie, bei der trotz der Stopp-Mutation "weitergelesen" werden soll, basiert auf der Entdeckung, dass winzige Moleküle (genannt TRIDs) diesen Abbruch durch das Stopp-Codon unterdrücken. Einige dieser TRIDs werden gegenwärtig untersucht und bei genetischen Störungen eingesetzt, die durch Nonsense-Mutationen verursacht wurden. Bis zum heutigen Zeitpunkt haben über 100 Studien die Wirksamkeit von TRIDs nachgewiesen.  Erkrankungen sind zum Beispiel: Mukoviszidose, Duchenne Muskeldystrophie, Louis-Bar-Syndrom und Lysosomale Speicherkrankheiten. Auch erbliche Augenerkrankungen wie z.B. Retinitis Pigmentosa, Kolobom, LCA, Usher und Aniridia wurden untersucht. Die Ergebnisse lassen auf die erfolgreiche Entwicklung von Therapien für diese Krankheiten hoffen.

ELX-02 der Firma Eloxx ist so ein TRID, und neben PTC124, der Firma PTC Therapeutics der nun zweite mir bekannte Wirkstoff, der es in klinische Studien geschafft hat.


The Foundation Fighting Blindness
berichtet über die START Therapie in Kanada,bei Nonsense Mutationen.

Wissenschaftler entwickeln Augentropfen zum Behandeln von erblichen Netzhauterkrankungen so berichtet aktuell die kanadische Foundation Fighting Blindness im Februar 2017 Ich berichtete schon früher in dieser Maillingliste über die pharmakologische Therapieoption bei Nonsense Mutationen und in diesem Kontext, passt die obige News Veröffentlichung zur Thematik. Für alle Interessierte möchte ich diese daher in Kürze zusammenfassen. Die sogenannte STAR-Therapie (Studie von Ataluren - des Herstellers PTC Therapeutics), versucht die Bildung eines funktionierenden Proteins zu ermöglichen, trotzt eines aufgrund einer Genmutation vorkommenden, biologischen Stopp Signals. Frau Dr. Gregory-Evans Forschungsinstitut und Universität Vancouver, hat sich hier auf die Erforschung von Augenerkrankungen, hervorgerufen durch Nonsense Mutationen spezialisiert. Die Forschung wird durch die Foundation Finding Blindness finanziell gefördert. Derzeit wird die Star Therapie bei der seltenen Augenerkrankung Anirida, in einer klinischen Studie erforscht. Der Erfolg in Mausmodellen, war so vielversprechend, der Weg zur klinischen Studie Phase 2 beschritten werden konnte. Die große Fragegestellung ob, Star auch für andere erbliche Netzhauterkrankungen in Frage kommt, ist die aktuelle Fragestellung und kommende Aufgabe des Forschungsteams. Als nächstes im Focus steht die Lebersche kongenitale Amaurose, da hier bei ungefähr 35 Prozent, eine Nonsense Mutation krankheitsverursachend vorliegt. Glücklicherweise haben die Forsche unterschiedliche Mausmodelle der LCA für vorklinische Studien bereit. Die Star Therapie verwendet zwei unterschiedliche Medikamente, Ataluren und Amlexanox, ein entzündungshemmender Wirkstoff der das Immunsystem beeinflussen kann. In der laufenden Studie werden Dosis und Mischungsverhältnis variiert eingesetzt, um herauszufinden was am besten wirkt u.a. auch beim RPE65 und RD3 Gen. Das Team arbeitet mit Hochdruck daran, auch bei anderen Netzhauterkrankungen explizit auch beim Usher Syndrom die Wirksamkeit zu testen. Neben Frau Dr. Mariya Moosajee, vom Moorfields Eye Hospital, ist Frau Dr. Gregory-Evans die zweite forschende Spezialistin auf diesem Gebiet der Augenheilkunde. In London sollte dieses Jahr ebenfalls ein Studie zu Nonsense Mutationen starten, und die Ergebnisse beider Studien werden Richtungsweisend sein. Grund genug die Therapieoption in den Focus zu stellen, und die Arbeiten zu verfolgen und zu unterstützen. Erste Forschungsergebnisse bestätigen, dass je früher eine Behandlung erfolgt, sich entsprechend bessere Ergebnisse zu erwarten sind. Eine molekulargenetische Diagnostik vorausgesetzt,  mit Bestimmung der genauen Mutationsart ist allerdings ein Vorraussetzung, um in Zukunft an weiteren Studien teilzunehmen. Das Patientenregister der Pro Retina, kann hier eine wegweisende Funktion der Studienteilnehmerakquirierung einnehmen. Bei Rückfragen zu Nonsense Mutationen oder weiteren Auskünfte stehe ich gerne zu Verfügung.

Externer Link: Original Artikel der kanadischen Foundation Finding Blindness http://ffb.ca/scientists-are-developing-an-eye-drop-to-treat-inherited-blinding-eye-diseases/

Externer Link: ClinicalTrials Link der Star Therapie http://clinicaltrials.gov/ct2/show/NCT02647359

Artikel zum Download
Treating Nonsense