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Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene.

Yoon SY, Gay-Antaki C, Ponde DE, Poptani H, Vite CH, Wolfe JH - Mol Ther Methods Clin Dev (2014)

Bottom Line: The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals.Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest.This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Children's Hospital of Philadelphia , Philadelphia, Pennsylvania, USA.

ABSTRACT
In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [(18)F]-fallypride analog of dopamine. The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

No MeSH data available.


Related in: MedlinePlus

Coexpression of D2R80A and green fluorescent protein (GFP) vectors in the mouse brain. Following positron emission tomography imaging, mice were euthanized for in situ hybridization and immunofluorescence to detect D2R80A mRNA, dopamine-2 receptor, GFP mRNA, and GFP in the: (a) hippocampus and thalamus and (b) cerebellum. The injection sites are shown with arrows on the D2R80A in situ hybridization sections.
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fig3: Coexpression of D2R80A and green fluorescent protein (GFP) vectors in the mouse brain. Following positron emission tomography imaging, mice were euthanized for in situ hybridization and immunofluorescence to detect D2R80A mRNA, dopamine-2 receptor, GFP mRNA, and GFP in the: (a) hippocampus and thalamus and (b) cerebellum. The injection sites are shown with arrows on the D2R80A in situ hybridization sections.

Mentions: Following PET imaging at the last time point, the mice were perfused and the brains were analyzed by in situ hybridization using D2R80A-specific probes and immunofluorescence using anti-D2R antibody. D2R80A expression seen by [18F]-fallypride binding in PET imaging correlated well with D2R80A mRNA and protein expression in all animals. Specific D2R80A expression was observed in the hippocampus and thalamus of the injected hemisphere, and the GFP-positive areas seen by in situ hybridization and fluorescence also correlated well with the PET imaging results (Figure 3a). Mice injected into the cerebellum showed exclusive D2R80A mRNA and protein expression in the injected hemisphere of the cerebellum, and this also correlated well with the GFP expression (Figure 3b).


Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene.

Yoon SY, Gay-Antaki C, Ponde DE, Poptani H, Vite CH, Wolfe JH - Mol Ther Methods Clin Dev (2014)

Coexpression of D2R80A and green fluorescent protein (GFP) vectors in the mouse brain. Following positron emission tomography imaging, mice were euthanized for in situ hybridization and immunofluorescence to detect D2R80A mRNA, dopamine-2 receptor, GFP mRNA, and GFP in the: (a) hippocampus and thalamus and (b) cerebellum. The injection sites are shown with arrows on the D2R80A in situ hybridization sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362377&req=5

fig3: Coexpression of D2R80A and green fluorescent protein (GFP) vectors in the mouse brain. Following positron emission tomography imaging, mice were euthanized for in situ hybridization and immunofluorescence to detect D2R80A mRNA, dopamine-2 receptor, GFP mRNA, and GFP in the: (a) hippocampus and thalamus and (b) cerebellum. The injection sites are shown with arrows on the D2R80A in situ hybridization sections.
Mentions: Following PET imaging at the last time point, the mice were perfused and the brains were analyzed by in situ hybridization using D2R80A-specific probes and immunofluorescence using anti-D2R antibody. D2R80A expression seen by [18F]-fallypride binding in PET imaging correlated well with D2R80A mRNA and protein expression in all animals. Specific D2R80A expression was observed in the hippocampus and thalamus of the injected hemisphere, and the GFP-positive areas seen by in situ hybridization and fluorescence also correlated well with the PET imaging results (Figure 3a). Mice injected into the cerebellum showed exclusive D2R80A mRNA and protein expression in the injected hemisphere of the cerebellum, and this also correlated well with the GFP expression (Figure 3b).

Bottom Line: The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals.Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest.This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Children's Hospital of Philadelphia , Philadelphia, Pennsylvania, USA.

ABSTRACT
In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [(18)F]-fallypride analog of dopamine. The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

No MeSH data available.


Related in: MedlinePlus