GTMB 7 - Gene Therapy & Molecular Biology

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Kairemo et al: Oligonucleotide radiotherapyTable II. Shows subcellular distributions calculated by the nucleus to nucleus absorbed dosesThe following SUVs at 20h after injection were given by Wu et al, 1999:6 mer 12 mer 20 mer 30 merKidney 53.1 13.3 17.8 1.9Liver 0.5 0.5 8.6 12.3Spleen 0.5 0.5 3.4 5.1The following subcellular distribution was assumed for 20 mer, approximately as in Wu et al, 1999:NucleusRestKidney 30% 70%Liver 30% 70%Tumour 80%, 50% 20%, 50%Table III shows the calculated absorbed doses for a number of organs and tumours.Macroscopic absorbed doses (mGy/MBq)Organ 111 In 90 Y 76 Br 211 AtLiver 0.63 4.41 1.29 4.90Spleen 0.43 3.59 0.96 1.91Kidney 0.95 10.1 2.35 8.74Whole body (mGy/MBq) 0.13 0.57 0.23 0.54Tumour, 100g 1.03 12.0 2.65 10.9Tumour, 0.01g 0.54 3.29 0.59 10.9Organ 111 In 90 Y 76 Br 211 AtLiver 0.63 4.41 1.29 4.90Table IV shows the cellular dosesAverage nucleus self-dose (mGy/MBq), and percentage of average nucleus absorbed doseOrgan 111 In 90 Y 76 Br 211 AtLiver 0.03 (4.0%) 0.004 (0.09%) 0.006 (0.5%) 0.21 (4.4%)Kidney 0.05 (4.8%) 0.007 (0.04%) 0.011 (0.5%) 0.38 (4.4%)Tumour, 100g 0.15 (14.5%), 0.09 0.023 (0.19%), 0.015 0.036 (1.4%), 0.023 1.26 (11.6%), 0.79Tumour, 20g 0.15 (27.8%), 0.09 0.023 (0.71%), 0.015 0.036 (6.1%), 0.023 1.26 (11.6%), 0.79Furthermore, ETS1 has expression in B and Tlymphocytes and thymus. Vascular endothelial growthfactor (VEGF) is an endothelial cell-specific mitogen thatpromotes angiogenesis in solid tumors. The VEGFinducedinvasiveness was inhibited by ETS1 antisenseoligonucleotides but not by a sense control (Chen 1997).Antisense-VEGF has been successfully used to controltumor growth and it may provide another basis for thedevelopment of antiangiogenic gene therapy (Saleh 1996).Rat glioma cells were transfected with a eukaryoticexpression vector bearing an antisense-VEGF cDNA andtransplanted into nude mice: growth of the antisense-VEGF cell lines was inhibited compared to control cells,despite the fact that they have a faster division time invitro. These tumors had fewer blood vessels and a higherdegree of necrosis explaining the reduced tumor size(Saleh 1996). Also, human melanoma cells transfectedwith sense vascular permeability factor (VPF)/VEGFexpressed and secreted large amounts of mouseVPF/VEGF and formed well-vascularized tumors withhyperpermeable blood vessels and minimal necrosis innude/SCID mice (Claffey, 1996).20
Gene Therapy and Molecular Biology Vol 7, page 21VPF/VEGF promoted melanoma growth bystimulating angiogenesis and constitutive VPF/VEGFexpression dramatically promoted tumor colonization inthe lung up to 50-fold of that of controls (Claffey, 1996).Minimal sequence information required for high-affinitybinding to VEGF is contained in 29-36-nucleotide motifsfor the development of potent and specific VEGFantagonists (Jellinek, 1994). Transforming growth factoralpha (TGF-alpha) has been shown to induce VEGF/VPFin normal human epidermal keratinocytes in vitro (Smyth,1997). By using a 19-mer antisense phosphorothioateoligodeoxynucleotide complementary to bases 6-24relative to the translational start site of the VEGF/VPFmRNA, modulation of VEGF/VPF induction by TGFalphawas examined in vitro. The anti-sense oligo wascapable of inhibiting VEGF/VPF RNA and protein tonear-basal levels providing an antiangiogenetic strategy(Smyth, 1997).Previously, it was shown that phosphorothioateantisense oligonucleotides directed against basic fibroblastgrowth factor (bFGF) mRNA inhibited both the growth ofKaposi's sarcoma (KS) cells derived from differentpatients and the angiogenic activity associated with thesecells, including the induction of KS-like lesions in nudemice (Ensoli, 1994). These effects were due to the blockof the production of bFGF which is required by AIDS-KScells to enter the cell cycle and which, after release,mediates angiogenesis (Ensoli, 1994).We describe oligos to be superior to peptide oligos invehicle characteristics of radiation. Whilephosphorothioate oligos have rapid disappearance fromplasma within an hour, and a biexponential elimination,their half lives apparently longer than in the peptideoligos. Although a phosphorothioate oligo leaves plasmarapidly, it requires days to leave the whole body. There isalso significant extravascular accumulation of greater than50 % of the injected dose over a period of 3 to 12 hr.Furthermore, uptake into tissues is not saturated, as someuptake is happening even at 28 days during continuosinfusion (Iversen et al, 1994). The oligos are extensivelyeliminated in the urine over first 3 days after bolusinjection. Distribution to, and tissue accumulation anddistribution is tissue-specific (Iversen et al, 1992, 1994).It can be addressed that the behavior of the radiationat small distancies is crucial. This would be crucial inoligoradiotherapy with highest possible uptake in thetarget cell and minimal radiation toxicity to surroundingnormal cells. Here, oligos are transferring radioactivesource inside the cell and finally to close contact withtarget RNA macromolecule.We have shown earlier that for subcellular targetinginternal labels give the lowest variation in estimatedabsorbed nuclear doses in our cell model with givendimensions (nuclear diameter 6-16 µm, cellular diameter12-20 µm) (Kairemo et al, 1996). From the published data(Crooke et al, 1995) for ISIS 2105,21-mer oligonucleotidethe following subcellular distribution was obtained: thenuclearuptake 0.2 %, cytoplasmic uptake 1.3 %, and cellsurface uptake 0.3 % of injected dose. In this anti-humanpapilloma virus (HPV) model these uptakes as % cellvolume are 11 % for nucleus, 72% for cytoplasm and 17% for cell surface. We calculated concentrationdistributions including the uniform distribution andpublished biodistribution. We normalized the resultsrelative to the uniform distribution and the effect of theactivity outside the cell was not taken into account, whichassumption lead to the maximal possible inhomogeneity inabsorbed dose distribution within a single cell.We have also calculated in vivo subcellular tissuedistribution for oligodeoxynucleotide phosphorothioateswith some Auger emitting radionuclides. Augeremittersare low-range electrons with high biologicalefficiency with a tendency of becoming more and morefrequently used, at least theoretically. The doses varyconsiderably depending on cellular dimensions whenusing Auger-emitting isotopes; however, in small cellsthey may give a high dose. In tumors cell dimensions mayvary and therefore these Auger-emitting isotopes shouldbe applied only when nuclear target circumstances arewell characterized. High energy β-emitter P-32 gives thenuclear dose closest to uniform distribution in cell sizes,but this is due to high energy. We have previously shown(Kairemo) that when using P-32 labelled oligos other thantarget cells will be destroyed because of long range. Thisis not the case when using β-emitters, P-33 and S-35,which are optimal when targets are smaller than 300 µm indiameter. P-33 was not studied here separately because itscharacteristics are very close to those of S-35. Now wealso demonstrate that calculations related to positronemitters F-18 and Br-76, beta-emitter Y-90, Auger-emitterIn-111 and alpha-emitter At-211 add substantialinformation to radionanotargeting dosimetry. Calculationsusing Br-76 demonstrate up to 5-fold differences in cellnuclear dose only in different cellular dimensions. Thisindicates the importanc of careful selection of a properradionuclide.It is possible to use a mixture of radioisotopes toensure a complete coverage of targets in more than onelocations, e.g. targeting nuclear related and cellular RNAat the same time. In addition, modern imaging techniqueallows visual control over kinetic events. Dual labellingmay provide therapeutic benefits when treating smallerand larger targets simultaneously. Further in vivodevelopment, especially with various labels for oligos ishighly indicated.ReferencesAgrawal S, Temsamani J, Galbraith W, Tang J. (1995)Pharmacokinetics of antisense oligonucleotides. ClinPharmacokinet 28, 7-16.Agrawal S, Temsamani J, Tang JY. (1991) Pharmacokinetics,biodistribution and stability of oligodeoxynucleotidephosphorothioates in mice. Proc Natl Acad Sci USA 88,7595-7599.Bolch WE, Bouchet LG, Robertson JS, Wessels BW, Siegel JA,Howell RW, Erdi AK, Aydogan B, Costes S, Watson EE,Brill AB, Charkes ND, Fisher DR, Hays MT, Thomas SR.(1999) MIRD pamphlet No. 17, the dosimetry of nonuniformactivity distributions--radionuclide S values at the voxel21
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