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- Chtarto, Abdelwahed, et al.
(author)
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A regulatable AAV vector mediating GDNF biological effects at clinically-approved sub-antimicrobial doxycycline doses
- 2016
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In: Molecular Therapy - Methods and Clinical Development. - : Elsevier BV. - 2329-0501. ; 5
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Journal article (peer-reviewed)abstract
- Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.
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| 3. |
- Kamel, Wael, 1986-
(author)
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Adenoviral small non-coding RNAs : A Structural and Functional Charaterization
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Since their discovery in 1953, adenoviruses have significantly contributed to the understanding of virus-host cell interactions, including mechanistic details of cellular processes such as cell cycle control and alternative RNA splicing. Among the first characterized adenoviral genes were the virus-associated RNAs (VA RNAI/II), which are produced in massive amount during a lytic infection. The VA RNAs perform multiple functions and are required for a successful adenovirus life cycle. More recently, it was shown that the VA RNAs are processed into small viral miRNAs, so-called mivaRNAs, which interfere with the function of the cellular RNAi/miRNA machinery.In papers I and II, we focused on a structural and functional characterization of the mivaRNAs using two approaches. Firstly, we created a model system where the predicted miRNA-like function of mivaRNAI could be investigated, without interfering with other VA RNA functions. This was accomplished by construction of recombinant adenoviruses, in which the seed sequence of mivaRNAI was altered. The results showed that in cell culture experiments the mivaRNAI seed sequence mutants grew as the wild type virus, suggesting that the mivaRNAs are not required during the lytic phase of an adenovirus infection. Secondly, we showed that the VA RNAs from different human adenoviruses (Ad4, Ad5, Ad11 and Ad37) undergo the same type of Dicer-dependent processing into mivaRNAs, which subsequently are loaded onto the RNA induced silencing complex (RISC), albeit with different efficiencies.In paper III, we demonstrated that the promoter proximal region of the adenovirus major late promoter (MLP) produces a novel non-canonical class of small RNAs, which we termed the MLP-TSS-sRNAs. Surprisingly the MLP-TSS-sRNA maintains the m7G-cap structure while bound to Ago2 containing RISC. These complexes are functional suppressing expression of target mRNAs with complementary binding site. Most importantly, the MLP-TSS-sRNA limits the efficiency of viral DNA replication probably through a targeting of the E2B mRNAs, which are transcribed in the antisense orientation. In conclusion, the MLP-TSS-sRNA represents the first viral small RNA, which has been shown to have a function as a regulator of an adenovirus infection.
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