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Scientific Paper Review
The authors of this paper describe their promising gene therapy technique for the treatment of LDL-receptor deficiency in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. The WHHL strain of rabbits was purposely developed with a mutation in the LDLR gene that essentially mimics the symptoms of familial hypercholesterolemia (FH) in humans (2). Using retroviral transplantation of the wild type LDLR gene, the WHHL rabbits showed about a 30% decrease in blood cholesterol levels.
This paper is fairly unique in that the sections (introduction, results, discussion, methods) are not specifically defined, however the authors smoothly transition from one section to another anyway.
To begin, the authors briefly explain the potential of gene therapy treatment and give a background on FH. The authors summarize the genetics, treatment, and symptoms of FH within one paragraph, requiring the reader to have some background knowledge of the disease. Next, previous successful studies on retroviral transplantation and WHHL rabbits are mentioned in order to support their reasons for this research. A brief explanation of the methodology of the development of the retroviral clone containing the wild type LDLR gene that was used in this study was included before the results.
The next part of the paper focused on how the wild type LDLR gene was implanted within WHHL rabbits. Because the gene therapy method is a very important aspect of the purpose of this study, it is explained in detail. By removing part of the WHHL rabbit liver and treating with collagenase, individual liver cells (hepatocytes) were obtained. These cells were then cultured and introduced with either the LDLR retrovirus or a control, a lacZ retrovirus. After transduction, the cells were injected into the spleen of the original rabbit, and most of the hepatocytes were transported to the liver. The researchers confirmed the presence of functioning LDL-receptors in the LDLR retroviral hepatocytes with fluorescent-labeled LDL and fluorescent microscopy. This was further quantified using an RNase assay and gel electrophoresis (Fig 1A from paper).
Because the researchers were interested in finding a long-term reduction in blood cholesterol levels, they periodically killed rabbits that had received the retroviral transplantations and tested for continued LDLR gene expression. They showed that the gene expression remained relatively constant for at least 6.5 months after the initial transplantation (Fig 1B from paper). This stability of retroviral gene expression over time is very promising from a human treatment standpoint. If successful in humans, repeated treatment would be unnecessary, which is a disadvantage of LDL apheresis, and entire liver transplantation could be bypassed.
The researchers also showed that some of the transplanted hepatocytes did remain in the spleen of the rabbits. An albumin gene, specifically expressed in hepatocyte cells, was probed using anti-sense RNA. Retroviral transplanted WHHL rabbits showed expression of the albumin gene, while non-transplanted rabbits did not (Figure 2 from paper). This further supports the effective implantation of the retroviral-transduced hepatocyte cells into WHHL rabbits.
Finally, the major result of this paper was the significant reduction of blood cholesterol levels in WHHL rabbits transplanted with retroviral LDLR. Over time (60 or 120 days) the cholesterol level in five rabbits declined an average of 308 mg/dL, or 25 to 45%, depending on the rabbit. The control rabbits showed no significant reductions in blood cholesterol levels (Figure 3 from paper).
This paper is fairly unique in that the sections (introduction, results, discussion, methods) are not specifically defined, however the authors smoothly transition from one section to another anyway.
To begin, the authors briefly explain the potential of gene therapy treatment and give a background on FH. The authors summarize the genetics, treatment, and symptoms of FH within one paragraph, requiring the reader to have some background knowledge of the disease. Next, previous successful studies on retroviral transplantation and WHHL rabbits are mentioned in order to support their reasons for this research. A brief explanation of the methodology of the development of the retroviral clone containing the wild type LDLR gene that was used in this study was included before the results.
The next part of the paper focused on how the wild type LDLR gene was implanted within WHHL rabbits. Because the gene therapy method is a very important aspect of the purpose of this study, it is explained in detail. By removing part of the WHHL rabbit liver and treating with collagenase, individual liver cells (hepatocytes) were obtained. These cells were then cultured and introduced with either the LDLR retrovirus or a control, a lacZ retrovirus. After transduction, the cells were injected into the spleen of the original rabbit, and most of the hepatocytes were transported to the liver. The researchers confirmed the presence of functioning LDL-receptors in the LDLR retroviral hepatocytes with fluorescent-labeled LDL and fluorescent microscopy. This was further quantified using an RNase assay and gel electrophoresis (Fig 1A from paper).
Because the researchers were interested in finding a long-term reduction in blood cholesterol levels, they periodically killed rabbits that had received the retroviral transplantations and tested for continued LDLR gene expression. They showed that the gene expression remained relatively constant for at least 6.5 months after the initial transplantation (Fig 1B from paper). This stability of retroviral gene expression over time is very promising from a human treatment standpoint. If successful in humans, repeated treatment would be unnecessary, which is a disadvantage of LDL apheresis, and entire liver transplantation could be bypassed.
The researchers also showed that some of the transplanted hepatocytes did remain in the spleen of the rabbits. An albumin gene, specifically expressed in hepatocyte cells, was probed using anti-sense RNA. Retroviral transplanted WHHL rabbits showed expression of the albumin gene, while non-transplanted rabbits did not (Figure 2 from paper). This further supports the effective implantation of the retroviral-transduced hepatocyte cells into WHHL rabbits.
Finally, the major result of this paper was the significant reduction of blood cholesterol levels in WHHL rabbits transplanted with retroviral LDLR. Over time (60 or 120 days) the cholesterol level in five rabbits declined an average of 308 mg/dL, or 25 to 45%, depending on the rabbit. The control rabbits showed no significant reductions in blood cholesterol levels (Figure 3 from paper).
Figure 1- The top graph shows the blood cholesterol levels over time of the lacZ control rabbits. The bottom graph shows the blood cholesterol levels over time of the LDLR retroviral rabbits. The LDLR rabbits show a significant decrease in blood cholesterol levels compared to the control rabbits. This was the major result of the paper. This image was taken from the reviewed paper and the reference can be found at the bottom of the page (1).
The authors go on to discuss the implications for this gene therapy treatment in humans. They understand that rabbits are metabolically different from humans, but this treatment is still feasible and should be considered for humans. Another important point noted is that a previous study tested for an antibody response to retroviral transplantation. It was shown that this was not the case in rabbits. Ideally, humans could also enjoy no immune response from this treatment.
This paper highlighted the importance of this research through effective results and in depth methodology. Much of the paper focused on proving that the retroviral LDLR hepatocytes were, in fact, expressing the correct protein, in the correct place, and over time. Although the most exciting result of the paper is the significant reduction in blood cholesterol in WHHL rabbits, the authors had to spend ample time proving the hepatocyte transplantation was a success because of the potential for this treatment to be used on humans. Even today, gene therapy is a controversial subject, and many people need to be convinced that it can potentially treat disease. At the time of publishing (1991), even more skepticism was sure to be surrounding gene therapy. The authors prove the success of the retroviral transplant, while also delivering exciting results directly connected to the transplant.
This paper highlighted the importance of this research through effective results and in depth methodology. Much of the paper focused on proving that the retroviral LDLR hepatocytes were, in fact, expressing the correct protein, in the correct place, and over time. Although the most exciting result of the paper is the significant reduction in blood cholesterol in WHHL rabbits, the authors had to spend ample time proving the hepatocyte transplantation was a success because of the potential for this treatment to be used on humans. Even today, gene therapy is a controversial subject, and many people need to be convinced that it can potentially treat disease. At the time of publishing (1991), even more skepticism was sure to be surrounding gene therapy. The authors prove the success of the retroviral transplant, while also delivering exciting results directly connected to the transplant.
References
1. Chowdhury, J. R., Grossman, M., Gupta, S., Chowdhury, N. R., Baker, J. R., & Wilson, J. M. (1991). Long-Term Improvement of Hypercholesterolemia After ex Vivo Gene Therapy in LDLR-Deficient Rabbits. Science, 254(5039), 1802-1805. doi: 10.1126/science.1722351
2. Buja, L. M., Kita, T., Goldstein, J. L., Watanabe, Y., & Brown, M. S. (1983). Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia. Arteriosclerosis, 3(1), 87-101. PMID: 6824499
2. Buja, L. M., Kita, T., Goldstein, J. L., Watanabe, Y., & Brown, M. S. (1983). Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia. Arteriosclerosis, 3(1), 87-101. PMID: 6824499
