Vehicles And Transduced Cells

The list is quite long for the array of vehicles being developed, both viral and nonviral, for the delivery of gene vectors. The details of such carriers are, of course, provided elsewhere throughout this book. There are 2 fundamentally different ways to track the biodistribution of delivered agents. One method is to track the sites of successful gene transfer/expression (various methods described in this chapter) and the other method relies on directly labeling vehicles, DNA or cells with radionuclides, fluorescent dyes, or MR-compatible contrast agents. The latter method may give better spatiotemporal information with respect to distribution of the vehicles or DNA material used for therapy, but does not give any information with regards to the success of gene transfer. The latter method has been reviewed recently and includes the direct labeling of herpes virus with 111In, adenoviral knob with 99mTc, liposomes with 111In, double-stranded DNA by peptide-based chelates ([99mTc]PBC), genetically modified mesothelioma cells with technitium ([99mTc]PA1-STK), myoblasts with techne-tium, and DNA-delivery systems with MRI-detectable, DNA-binding chelates (86-93).

For those involved in the development of novel viral or nonviral delivery vehicles, targeting and efficiency of gene transfer are primary concerns, and, thus, examples of the former method are given here. The success of exogenous gene expression is dependent on its ability to at least survive the following series of stringent events: the DNA-vehicle complex has to bind specific cell-surface receptors, undergo receptor-mediated endocytosis, survive endosomal lysis, be released from endosomal captivity, endure the cytoplasmic environment, be destined for targeted entry of the nucleus, and ultimately released from carrier molecules to facilitate gene expression (94). Whether it is pseudo-typed lentiviruses, modified PEI complexes, liposomes, etc., one major role reporter genes are expected to provide is the monitoring of localization, biodistribution, and gene transfer efficiency of these delivery vehicles in living subjects. Noninvasive localization

Figure 12 Magnetic Resonance Spectroscopy (MRS) of Transgene Expression 31P-MRS has the ability to detect phosphorus NMR signals. A recombinant adenovirus (rAdCMVak) can be constructed to deliver arginine kinase, an enzyme unique to invertebrates, into muscle. Once introduced into mammalian muscle, the enzyme catalyzes the production of phosphoarginine (PArg), which has a unique spectral signature (both in magnitude and location on the spectrum) that can be detected by MRS. Figure represents in vivo basal 31P spectra from the hind limbs of a 6-month-old mouse. 31P-MRS spectra from the rAdCMVAK-injected limb (upper spectrum) reveal a 31P resonance at the chemical shift for PArg that is not present in the contralateral control limb (lower spectrum). (Images reproduced with permission from Ref. 84.)

Figure 12 Magnetic Resonance Spectroscopy (MRS) of Transgene Expression 31P-MRS has the ability to detect phosphorus NMR signals. A recombinant adenovirus (rAdCMVak) can be constructed to deliver arginine kinase, an enzyme unique to invertebrates, into muscle. Once introduced into mammalian muscle, the enzyme catalyzes the production of phosphoarginine (PArg), which has a unique spectral signature (both in magnitude and location on the spectrum) that can be detected by MRS. Figure represents in vivo basal 31P spectra from the hind limbs of a 6-month-old mouse. 31P-MRS spectra from the rAdCMVAK-injected limb (upper spectrum) reveal a 31P resonance at the chemical shift for PArg that is not present in the contralateral control limb (lower spectrum). (Images reproduced with permission from Ref. 84.)

of retroviral- (95), adenoviral- (96), and herpes viral vector-(97) mediated HSV1-ifc gene transfer has been performed. Fig. 13 is an example of replication-conditional, oncolytic herpes simplex virus-mediated gene delivery. Constitutively expressed optical and/or PET reporter genes have been helpful in studying the distribution of nonviral vehicles such as PEI polyplexes, which have been covalently modified with transferrin to facilitate targeting (Fig. 14), and cationic lipid-DNA complexes (Fig. 15).

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