Use of protamine to augment adenovirus-mediated cancer gene therapy

Our plain-English summary of Use of protamine to augment adenovirus-mediated cancer gene therapy, by M. Lanuti, C.E. Kouri, S. Force, et al (Gene Therapy, 1999).

The study

• Authors: M. Lanuti, C.E. Kouri, S. Force, M. Chang, K. Amin, K. Xu, I. Blair, L. Kaiser, S. Albelda
• Journal: Gene Therapy
• Published: 1999;6(9):1600-10
• Cited by: 44 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Improving the therapeutic potential of adenoviral (Ad) suicide gene therapy has become an area of intense investigation since the inception of gene therapy strategies for cancer treatment. Poor efficiency of gene transfer to target tissues has become one of the most important limitations to Ad-based gene therapy. Since polycations have been shown to enhance adenovirus-mediated gene transfer in epithelial cells both in vitro and in vivo, we hypothesized that polycations could augment treatment efficacy in animals with established tumor. To address this hypothesis, protamine sulfate, a polycation already safely administered in humans, was complexed with a recombinant Ad (E1E3-deleted) vector containing the herpes simplex 1 thymidine kinase (HSVtk) suicide gene to treat cancer cell lines in vitro and in animals bearing intraperitoneal tumor. In the presence of 5 microg/ml protamine, the efficiency of gene transfer to a number of cancer cell lines normally resistant to adenovirus was significantly enhanced. Protamine's effect in vitro was found to be inversely proportional to the level of expression of the high affinity Ad binding site, coxsackievirus and adenovirus receptor (CAR), on the sur- face of the various cell lines tested. Ad.tk infected tumor cells were rendered 2.5- to three-fold more sensitive to 20 microM ganciclovir (GCV) in the presence of protamine. Protamine also augmented the in vivo transfer efficiency of the marker gene, LacZ (contained in an Ad vector), on the surface of tumors derived from an intraperitoneal mouse model. Quantitative imaging revealed 50% tumor surface transduced with LacZ when treatment was performed in the presence of 50 microg/ml protamine compared with 12% tumor surface in controls. However, experiments performed utilizing intraperitoneal administration of Ad.tk/GCV in the presence or absence of 50 microg/ml protamine demonstrated no significantly improved median survival in mice bearing established intraperitoneal tumors. Similarly, in Fischer rats bearing intrapleural tumor, no improvement in anti-tumor response was observed when Ad treatment was performed intrapleurally in the presence of protamine. Thus, although protamine induced an enhancement of Ad-mediated gene transfer in vitro and in vivo, its use as an adjunct to intracavitary Ad-based cancer gene therapy in vivo appears to be limited.

In plain English

This was a laboratory and animal study testing whether the polycation protamine sulfate improves adenovirus (Ad)-mediated gene transfer and suicide gene therapy for cancer. Eight cell lines, including the human mesothelioma lines REN, I-45 and H2052 and the rat mesothelioma line II-45 (derived from asbestos-induced mesothelioma in F344 rats), were infected with Ad vectors with or without protamine; surface expression of the adenovirus receptor CAR could be measured directly only in the human cell lines, because the antibody did not cross-react with rat or mouse cells. Protamine lowered the dose of virus needed to infect half the cells by 22-, 11-, nine- and 4.5-fold in EJ-62, H2052, II-45 and HASMC cells, but reduced infectability two-fold in the highly infectable REN line. Protamine increased suicide gene killing in vitro and raised LacZ staining of intraperitoneal mouse tumour surfaces from 12% to 50%. However, it did not improve median survival in tumour-bearing mice, and in Fischer rats with intrapleural mesothelioma no statistically significant further reduction in tumour weight was detected. The authors concluded protamine's use as an adjunct to intracavitary Ad-based cancer gene therapy in vivo appears limited.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Experimental laboratory study: in vitro cell culture experiments plus in vivo experiments in mice (intraperitoneal tumour) and Fischer rats (intrapleural mesothelioma)
• Who was studied: Eight cultured cell lines (including human mesothelioma REN, I-45, H2052 and rat mesothelioma II-45, plus HeLa as reference); BALB/c and SCID mice bearing intraperitoneal EJ-62 tumours (n = 9 per group in the survival study, n = 3 per group for LacZ imaging); Fischer 344 rats bearing intrapleural II-45 mesothelioma (n = 10 per group, repeated three times), University of Pennsylvania laboratories
• Exposure: Protamine sulfate (doses from 0.5 to 500 micrograms per millilitre; most in vitro work at 5 micrograms per millilitre, animal work at 50 micrograms per millilitre) given with recombinant E1E3-deleted adenoviral vectors carrying PECAM-1, LacZ, cytosine deaminase or HSV thymidine kinase, with the prodrugs 5-fluorocytosine or ganciclovir
• Main results:
  • Protamine reduced the viral dose needed to infect 50% of cells by 22-fold (EJ-62), 11-fold (H2052), nine-fold (II-45) and 4.5-fold (HASMC); smaller effects of 1.9-, 1.3- and 1.2-fold in A549, H-322 and I-45
  • In the highly infectable REN human mesothelioma line, protamine reduced rather than increased infectability
  • The authors reported the protamine effect was inversely proportional to cell-surface hCAR expression (relative to HeLa = 100%: REN 152.3%, I-45 55.6%, H-322 36.0%, A549 28.7%, HASMC 4.7%, H2052 0.7%); hCAR could not be measured in the rat and mouse lines
  • With protamine, II-45 cells infected with Ad.CMVcd at 100 MOI were 25-fold more sensitive to 5-fluorocytosine
  • With protamine, Ad.RSVtk-infected EJ-62 cells were 10-fold more sensitive to ganciclovir (and II-45 cytotoxicity increased five-fold)
  • Protamine did not change intracellular ganciclovir levels in II-45 cells: 0.007 ± 0.001 pg per cell with 5 micrograms per millilitre protamine versus 0.008 ± 0.0012 pg per cell without
  • In mice given intraperitoneal Ad.CMVlacZ, 12% of tumour surface stained without protamine, rising to 30% (5 micrograms per millilitre, P = 0.0018) and 50% (50 micrograms per millilitre, P less than 0.0001); 500 micrograms per millilitre gave about 10%
  • Surface staining increased with protamine at the two lower doses
  • In tumour-bearing mice, adding protamine to Ad.RSVtk plus ganciclovir gave no median survival benefit (about 38 days in BALB/c, about 33 days in SCID)
  • In Fischer rats with intrapleural II-45 mesothelioma, tumour weight was 3.66 ± 0.17 g in controls, 1.79 ± 0.2 g with virus and 1.62 ± 0.15 g with virus plus protamine; the difference between the two treated groups was not statistically significant (P = 0.5658)
• The authors' conclusion: The authors concluded that although protamine enhanced adenovirus-mediated gene transfer in vitro and on tumour surfaces in vivo, it did not improve anti-tumour effects, so its use as an adjunct to intracavitary adenoviral cancer gene therapy in vivo appears limited; they suggested the enhanced surface expression might still be useful in other gene therapy settings
• Limitations the authors state:
  • The authors stated the reason for the lack of improved efficacy in vivo was unknown, and hypothesised that protamine enhanced only surface transfection without increasing viral penetration into solid tumour
  • CAR expression could not be measured directly in the rat (II-45) and mouse (EJ-62) cell lines because the antibody did not cross-react
  • The authors noted the full in vivo effect of protamine may have been impaired by intracavitary serum
  • The mechanism by which polycations increase gene transfer was not well understood

Topics

Medical terms in this study, explained in our Lexicon: Median Survival.

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• Gene therapy using adenovirus carrying the herpes simplex-thymidine kinase gene to treat in vivo models of human malignant mesothelioma and lung cancer — H.C. Hwang, W.R. Smythe, et al. (1995)
• Chemotherapy delivered after viral immunogene therapy augments antitumor efficacy via multiple immune-mediated mechanisms — Z.G. Fridlender, J. Sun, et al. (2010)
• Efficacy of repeated adenoviral suicide gene therapy in a localized murine tumor model — E.S. Lambright, S.D. Force, et al. (2000)
• Inhibition of mesothelioma cancer stem-like cells with adenovirus-mediated NK4 gene therapy — X.B. Deng, L. Xiao, et al. (2015)
• Imaging in vivo herpes simplex virus thymidine kinase gene transfer to tumour-bearing rodents using positron emission tomography and — R. Hustinx, C.Y. Shiue, et al. (2001)

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