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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

Serial positron emission tomography (PET) imaging following intraparenchymal injection of AAV1.D2R80A in adult mice. (a) Adult mice (n = 6) were coinjected unilaterally with equivalent titers (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum of the left hemisphere. (b) A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in the left hemisphere and the cerebellum in the right hemisphere. Serial PET studies were performed in both groups monthly up for 3 months postinjection. PET signal was measured in each animal at each time point by quantifying [18F]-fallypride binding relative to the striatum in: (c) the cerebellum following a single unilateral injection and (d) the hippocampus and thalamus and (e) the cerebellum following multiple injections in the second group of mice. AAV, adeno-associated virus; Cer, cerebellum; GFP, green fluorescent protein; HG, harderian gland; hp, hippocampus; L, left hemisphere; R, right hemisphere; str, striatum; th, thalamus.
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fig2: Serial positron emission tomography (PET) imaging following intraparenchymal injection of AAV1.D2R80A in adult mice. (a) Adult mice (n = 6) were coinjected unilaterally with equivalent titers (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum of the left hemisphere. (b) A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in the left hemisphere and the cerebellum in the right hemisphere. Serial PET studies were performed in both groups monthly up for 3 months postinjection. PET signal was measured in each animal at each time point by quantifying [18F]-fallypride binding relative to the striatum in: (c) the cerebellum following a single unilateral injection and (d) the hippocampus and thalamus and (e) the cerebellum following multiple injections in the second group of mice. AAV, adeno-associated virus; Cer, cerebellum; GFP, green fluorescent protein; HG, harderian gland; hp, hippocampus; L, left hemisphere; R, right hemisphere; str, striatum; th, thalamus.

Mentions: Adult mice (n = 6) were coinjected unilaterally with equivalent doses (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum at a site distal to the striatum where there is no endogenous D2R expressed. A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in one hemisphere and the cerebellum in the opposite hemisphere. Serial PET studies were performed monthly for up to 3 months postinjection in the same animals to assess persistence of gene expression and detection by [18F]-fallypride binding. In the first group of mice, imaging indicated [18F]-fallypride accumulation in the cerebellum in the injected hemisphere, whereas [18F]-fallypride binding was only detected in the striatum of the uninjected mouse brain (Figure 2a). The sagittal view of the image also shows radioactivity in the harderian glands (located in the orbital cavity), which accumulates tracers nonspecifically.35–37 Radiotracer accumulates nonspecifically outside the brain in the harderian glands, salivary glands, nasal cavity, and orbital cavity as well as the spinal column.


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)

Serial positron emission tomography (PET) imaging following intraparenchymal injection of AAV1.D2R80A in adult mice. (a) Adult mice (n = 6) were coinjected unilaterally with equivalent titers (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum of the left hemisphere. (b) A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in the left hemisphere and the cerebellum in the right hemisphere. Serial PET studies were performed in both groups monthly up for 3 months postinjection. PET signal was measured in each animal at each time point by quantifying [18F]-fallypride binding relative to the striatum in: (c) the cerebellum following a single unilateral injection and (d) the hippocampus and thalamus and (e) the cerebellum following multiple injections in the second group of mice. AAV, adeno-associated virus; Cer, cerebellum; GFP, green fluorescent protein; HG, harderian gland; hp, hippocampus; L, left hemisphere; R, right hemisphere; str, striatum; th, thalamus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Serial positron emission tomography (PET) imaging following intraparenchymal injection of AAV1.D2R80A in adult mice. (a) Adult mice (n = 6) were coinjected unilaterally with equivalent titers (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum of the left hemisphere. (b) A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in the left hemisphere and the cerebellum in the right hemisphere. Serial PET studies were performed in both groups monthly up for 3 months postinjection. PET signal was measured in each animal at each time point by quantifying [18F]-fallypride binding relative to the striatum in: (c) the cerebellum following a single unilateral injection and (d) the hippocampus and thalamus and (e) the cerebellum following multiple injections in the second group of mice. AAV, adeno-associated virus; Cer, cerebellum; GFP, green fluorescent protein; HG, harderian gland; hp, hippocampus; L, left hemisphere; R, right hemisphere; str, striatum; th, thalamus.
Mentions: Adult mice (n = 6) were coinjected unilaterally with equivalent doses (1.5 × 1010 GC) of AAV1.D2R80A and AAV1.GFP into the cerebellum at a site distal to the striatum where there is no endogenous D2R expressed. A second group of mice (n = 4) was given multiple injections of AAV1.D2R80A and AAV1.GFP (2 × 1010 GC per site) into the hippocampus and thalamus in one hemisphere and the cerebellum in the opposite hemisphere. Serial PET studies were performed monthly for up to 3 months postinjection in the same animals to assess persistence of gene expression and detection by [18F]-fallypride binding. In the first group of mice, imaging indicated [18F]-fallypride accumulation in the cerebellum in the injected hemisphere, whereas [18F]-fallypride binding was only detected in the striatum of the uninjected mouse brain (Figure 2a). The sagittal view of the image also shows radioactivity in the harderian glands (located in the orbital cavity), which accumulates tracers nonspecifically.35–37 Radiotracer accumulates nonspecifically outside the brain in the harderian glands, salivary glands, nasal cavity, and orbital cavity as well as the spinal column.

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