Genetic Incorporation of Human Metallothionein into the Adenovirus Protein IX for Non-Invasive SPECT Imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of 99mTc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a 99mTc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo

Successful Treatment of Richter Transformation with Ibrutinib in a Patient with Chronic Lymphocytic Leukemia following Allogeneic Hematopoietic Stem Cell Transplant

Patients with chronic lymphocytic leukemia (CLL) who progress to Richter transformation (RT) have a poor prognosis. Multi-agent chemotherapy regimens do not have good response rates. There are few case reports on the use of ibrutinib in RT. Here, we present a patient who was heavily pretreated for CLL, including allogeneic stem cell transplant, and progressed to RT. She had a mixed response to multi-agent chemotherapy and was started on ibrutinib. She had a complete response for 16 months on single-agent ibrutinib with minimal toxicity

SPECT imaging analysis of a mouse injected with an adenovirus containing a capsid-incorporated pIX-MT fusion protein.

<p>C57BL6 mice were injected intravenously with approximately 1 mCi of ^99mTc in 0.3 mL PBS, and scanned at 30 min after the injection. Afterwards, 3D renderings of SPECT images in blue-purple-red-yellow scale against CT projections in grey scale were obtained. Shown are: (A) coronal image of a normal mouse injected with ^99mTc-pertechnetate; (B) coronal image of a mouse injected with ^99mTc-glucoheptonate; (C) coronal and sagittal images of a normal mouse injected with ^99mTc-Ad-tGFP-pIX-MT; and (D) coronal and sagittal images of a mouse pretreated with warfarin injected with ^99mTc-Ad-tGFP-pIX-MT.</p

Comparison of ^99mTc radiolabel stability on Ad-tGFP-pIX-MT.

<p>After 30 min incubation with 50% normal mouse serum at increasing temperatures, samples were analyzed by HPLC on a Bio-Monolith column for ^99mTc-radiolabeled Ad-tGFP-pIX-MT in each sample. Radioactivity of peaks corresponding to Ad-tGFP-pIX-MT were quantified and normalized to the radio activity of peaks corresponding to Ad-tGFP-pIX-MT after 30 min in the absence of serum at 27°C. Each point represents the mean ± SE of three replicate samples.</p

Concentration dependence of competition for metal binding to purified virus.

<p>Aliquots of ^99mTc-radiolabeled Ad-tGFP-pIX-MT were incubated for 15 min at 37°C with the indicated concentrations of CdCl₂, CoCl₂, CuCl₂, or ZnCl₂. Virus bound ^99mTc was determined counting radioactivity after purification through micro spin columns.</p

Gel filtration column profile of 99mTc binding to adenovirus containing a pIX-MT fusion protein.

<p>After incubation with^99mTc glucoheptonate, the Ad-tGFP-pIX-MT virus containing a pIX-MT fusion protein and the Ad-CMV-EGFP virus containing a wild-type pIX protein were purified by gravity gel filtration using 20 mL Sephacryl S-200 columns. Each point represents the specific activity in mBq/v.p of individual 10 drop fractions collected.</p

Construction and analysis of a pIX-MT containing adenovirus.

<p>(A) Schematic representation of pIX-MT and a CMV-tGFP expression cassette inserted into the Ad5 genome. (B) Expression of GFP in cells infected with Ad-tGFP-pIX-MT shows formation of an individual plaque. (C) Detection of adenovirus fiber and metallothionein proteins incorporated into the purified Ad-tGFP-pIX-MT and Ad-CMV-EGFP viral particles by western blot analysis.</p