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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

Positron emission tomography (PET) signal in the mouse cortex following bilateral intraventricular vector injection in neonatal mice. (a) Neonatal mice (n = 5) were injected bilaterally into the lateral ventricles with equivalent titers (9 × 109 GC) of AAV1.D2R80A and AAV.GFP and then imaged 4 weeks postinjection by PET with [18F]-fallypride. (b) Mean radioactivity in the cortex of each animal was measured by quantifying [18F]-fallypride binding in the cortex relative to binding in the striatum. AAV, adeno-associated virus; Ctx, cortex; GFP, green fluorescent protein; L, left hemisphere; R, right hemisphere; str, striatum; uninj, uninjected.
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fig1: Positron emission tomography (PET) signal in the mouse cortex following bilateral intraventricular vector injection in neonatal mice. (a) Neonatal mice (n = 5) were injected bilaterally into the lateral ventricles with equivalent titers (9 × 109 GC) of AAV1.D2R80A and AAV.GFP and then imaged 4 weeks postinjection by PET with [18F]-fallypride. (b) Mean radioactivity in the cortex of each animal was measured by quantifying [18F]-fallypride binding in the cortex relative to binding in the striatum. AAV, adeno-associated virus; Ctx, cortex; GFP, green fluorescent protein; L, left hemisphere; R, right hemisphere; str, striatum; uninj, uninjected.

Mentions: Intraventricular injection of neonatal mice with AAV1 results in widespread transduction of cells in the neocortex, the entorhinal cortex, and the hippocampus.33 Four weeks following neonatal injection with AAV1.D2R80A into the cerebral lateral ventricles, mice (n = 5) were imaged by PET with [18F]-fallypride to examine the binding pattern in the brain. PET imaging revealed [18F]-fallypride accumulation in the striatum where endogenous D2R is expressed, and throughout the cortical regions of the mouse brains (Figure 1a), indicating expression of D2R80A. In the transverse view, portions of the striatum and the cortex are superimposed, thus the [18F]-fallypride signal appear to overlap, but separation of the two regions is apparent in the coronal view.


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)

Positron emission tomography (PET) signal in the mouse cortex following bilateral intraventricular vector injection in neonatal mice. (a) Neonatal mice (n = 5) were injected bilaterally into the lateral ventricles with equivalent titers (9 × 109 GC) of AAV1.D2R80A and AAV.GFP and then imaged 4 weeks postinjection by PET with [18F]-fallypride. (b) Mean radioactivity in the cortex of each animal was measured by quantifying [18F]-fallypride binding in the cortex relative to binding in the striatum. AAV, adeno-associated virus; Ctx, cortex; GFP, green fluorescent protein; L, left hemisphere; R, right hemisphere; str, striatum; uninj, uninjected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Positron emission tomography (PET) signal in the mouse cortex following bilateral intraventricular vector injection in neonatal mice. (a) Neonatal mice (n = 5) were injected bilaterally into the lateral ventricles with equivalent titers (9 × 109 GC) of AAV1.D2R80A and AAV.GFP and then imaged 4 weeks postinjection by PET with [18F]-fallypride. (b) Mean radioactivity in the cortex of each animal was measured by quantifying [18F]-fallypride binding in the cortex relative to binding in the striatum. AAV, adeno-associated virus; Ctx, cortex; GFP, green fluorescent protein; L, left hemisphere; R, right hemisphere; str, striatum; uninj, uninjected.
Mentions: Intraventricular injection of neonatal mice with AAV1 results in widespread transduction of cells in the neocortex, the entorhinal cortex, and the hippocampus.33 Four weeks following neonatal injection with AAV1.D2R80A into the cerebral lateral ventricles, mice (n = 5) were imaged by PET with [18F]-fallypride to examine the binding pattern in the brain. PET imaging revealed [18F]-fallypride accumulation in the striatum where endogenous D2R is expressed, and throughout the cortical regions of the mouse brains (Figure 1a), indicating expression of D2R80A. In the transverse view, portions of the striatum and the cortex are superimposed, thus the [18F]-fallypride signal appear to overlap, but separation of the two regions is apparent in the coronal view.

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