© Springer Japan 2016
Shuji Terai and Takeshi Suda (eds.)Gene Therapy and Cell Therapy Through the Liver10.1007/978-4-431-55666-4_11. Liver-Targeted Gene and Cell Therapies: An Overview
(1)
Department of Pharmaceutical and Biomedical Sciences, University of Georgia College of Pharmacy, 250 W. Green Street, Athens, GA 30602, USA
Abstract
Until very recently, management of several liver malignancies, viral hepatitis, hepatic cirrhosis, and hereditary metabolic diseases remained unsatisfactory, and thus, efficient therapeutic approaches have always been in need. In parallel with recent advances in molecular biology and recombinant DNA technologies, research in liver diseases and the quest for molecular insights of disease pathology have witnessed remarkable progression, and early and specific detection of genetic, infectious, and malignant liver diseases has become feasible like never before. Several molecular approaches combining genetics, biology, chemistry, and computer sciences have been introduced, and in particular, gene- and cell-based therapies opened up new opportunities that step out beyond classical pharmacology. Gene therapy emerged as promising therapeutic strategy aiming to introduce genetic material into cells to generate curative effects. Gene therapy comprises various methods of gene delivery and innovative overexpression and silencing designs for specific therapeutic needs (Kay MA, Nat Rev Genet 12:316–328, 2011). Cell-based therapies, on the other hand, aim to use biologically active living cells instead of DNA or RNA as treatment modality. Several extracorporeal and implantable cell therapies have been developed, such as bioartificial liver (Baquerizo A, Mhoyan A, Kearns-Jonker M, Arnaout WS, Shackleton C, Busuttil RW et al, Transplantation 67:5–18, 1999) for short-term treatment and liver cell transplantation for permanent liver replacement (Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, Warkentin PI et al, N Engl J Med 338:1422–1426, 1998). In this chapter, we will briefly discuss the current theories and potential applications of gene- and cell-based therapies for the treatment and/or prevention of various liver diseases.
Keywords
Gene therapyLiver gene therapyCell therapyLiver diseaseGene delivery1.1 Gene Therapies
Liver diseases can be classified into inherited monogenic disorders caused by a single gene defect, such as liver cystic fibrosis, and multifactorial disorders caused by defects of several genes that are acquired and accumulated during lifetime, such as liver malignancies. Most inherited metabolic disorders have an underlying genetic defect in the liver [1–4]. Such defects often result in loss, dysfunction, or accumulation of proteins or metabolites contributing to disease pathology. Gene therapy has evolved as a potent means of treatment of pathogenic gene defects using nucleic acids to alter gene expression and restore pathogenic phenotype into normal status. Importantly, the potential of gene therapy is not limited to monogenic hereditary diseases but also for various multifactorial disorders, such as infections, degenerative diseases, and cancers.
1.1.1 Liver-Directed Gene Delivery
To elicit curative effects, therapeutic genes must be safely and efficiently introduced into liver cells. Importantly, the real target for liver-directed gene transfer is liver parenchymal cells (hepatocytes), which account for 65 % of liver resident cells, rather than sinusoidal cells which constitute the remaining fraction, including endothelial cells, Kupffer cells (liver macrophages), stellate cells or Ito cells, and pit cells (natural killer cells) [5]. Sinusoidal endothelial and Kupffer cells are functionalized scavenger cells for particulate entities and, thus, considered a major challenge for liver-directed gene transfer [6]. Diverse systems have been developed to overcome barriers of gene delivery utilizing biological systems, such as viral and bacterial systems, and non-biological systems, including chemical and physical methods of gene transfer [7]. To date, viral vectors, which are engineered replication-deficient viruses, remain the most efficient delivery systems and the most commonly used in clinic [8]. However, their immunogenicity and insertional mutagenesis are serious hurdles to be overcome. Several types of viruses have been utilized for liver-directed gene transfer, among which adeno-associated virus is the prime vector to be applied in clinic [9], as being highly efficient and nonpathogenic. Non-viral methods of gene transfer comprising synthetic vectors and physical methods evolved as safer alternatives to viral vectors. Yet, the efficiency of non-viral systems is currently a subject of intensive research to be optimized. Synthetic vectors implicate polymers and liposomes as carriers to protect genetic material from biodegradation and to facilitate cell entry via endocytosis [10]. Physical methods preclude the need of vectors by direct gene transfer into cells through physically created transient pores in cell membrane. Different mechanical forces have been applied for liver-directed gene transfer, such as electrical pulses, hydrodynamic pressure, ultrasound waves, laser, and particle bombardment [11]. Hydrodynamic gene transfer is the most commonly used non-viral method for liver-directed gene transfer because of high efficacy and recent advancement into lobe-specific computerized injection into the liver in rodents and large animals [12].
1.1.2 Gene Therapy Strategies for Liver Disease Treatment
Gene therapy is conducted through various approaches to overexpress or block the expression of certain genes depending on the type of gene defect.
1.1.2.1 Gene Replacement
Liver disorders can be corrected by replacing the defected or inactive gene with wild-type homologue. This approach holds promise for diseases with single gene defect, and indeed it was successfully employed for gene therapy of hemophilia [13] and hereditary tyrosinemia type I [14]. Similarly, gene replacement can be applied for multifactorial liver diseases that are nonetheless characterized by a single gene dysfunction, such as p53 in hepatocellular carcinoma (HCC) [15].
1.1.2.2 Gene Repair
Another approach to correct monogenic liver diseases is to repair defective genes in situ through utilization of innate homologous recombination cells commonly use to repair DNA breaks. The rationale is to use natural or recombinant nuclease enzymes that specifically recognize the mutated genetic sequence and create a DNA break. With proper method of gene transfer, DNA template for the desired functional sequence is provided in diseased cells, and the mutated fragment can be repaired by an exchange of genetic sequences through homologous recombination [16]. Mutated sequences can also be targeted by chimeric oligonucleotides specifically hybridizing mutated genes and successfully applied for gene therapy of Crigler-Najjar syndrome type I [17] and hyperlipidemia type III [18].
1.1.2.3 Gene Augmentation
Boosting gene expression for liver disease management is typically used when a given gene product is absent or insufficient to restore the normal physiologic status. Gene therapy of multifactorial liver diseases, such as HCC, is often explored with gene augmentation approach. Diverse strategies have been evolved for HCC gene therapy, such as suicide gene therapy relying on overexpressing a gene encoding a prodrug-activating enzyme in tumor tissue followed by prodrug administration to spare cytotoxic effects for malignant cells only [19]. Another form of cancer gene therapy is oncolytic viruses, where engineered replication-competent viruses selectively replicate in malignant cells, resulting in progressive destruction of tumor mass [20]. HCC treatment was also explored with antiangiogenic gene therapy using angiostatin gene [21] and immunotherapy to induce antitumor immune response by means of cytokine gene overexpression [22].
1.1.2.4 DNA Vaccination
Transferring genes coding for tumor-specific surface antigens or viral antigens to elicit durable humoral and cellular immune response has gained increased attention as therapeutic and preventive modalities for HCC and viral hepatitis. Indeed, DNA vaccination with alpha-fetoprotein-expressing plasmid DNA successfully inhibited growth of HCC in mice by triggering antitumor immune response [23]. Likewise, DNA vaccination with plasmid DNA-expressing hepatitis C virus (HCV) genotype 1a/1b proteins induced potent cell-mediated immune response to HCV in mice and nonhuman primates [24].
1.1.2.5 Gene Silencing
Blockade of specific gene expression or function that is thought to underlie disease pathology is another gene therapy approach to tackle disease progression. Increased expression of pathogenic proteins is often reported with acquired liver diseases like infections and cancers. Different strategies have been developed to silence gene expression to interfere with transcription, RNA transport and stability, and translation. Among these, ribozymes can inhibit gene expression and viral replication by catalyzing sequence specific RNA cleavage [25]. Ribozymes were successfully applied for in vitro inhibition of hepatitis B virus (HBV), but in vivo efficacy is yet to be approved [26, 27]. Oligonucleotides (ON) are also used for gene silencing purposes, and several designs have been introduced such as antisense ON and small interfering RNA molecules, both hybridize to specific regions in the target gene or its corresponding mRNA transcript, respectively. ONs are increasingly used to treat liver cancers [28, 29] and viral infections [30, 31]. Inhibition of gene function at protein level is also achievable using dominant negative mutant method, in which genes coding for nonfunctional peptides or proteins are delivered to diseased cells to interfere with their counterparts [32], and promising potential was demonstrated in the management of HBV infections [33, 34].
1.2 Cell Therapies
Liver transplantation has been the only effective treatment for end-stage liver diseases. However, because of the serious challenges such as shortage of organ donors and the consequences of long-term immune suppression after transplantation [35], alternative therapies are needed. Cell therapies have evolved as regenerative therapies relying on stem cell and tissue regeneration technologies to rebuild or regrow liver organ to provide the missing product. Theories of cell-based therapies in liver diseases are inspired by a broader understanding of mechanisms underlying the natural ability to regenerate organs in some model organisms in nature [36] and the mechanisms of liver regeneration and repair, including activation of local stem cells [37] and contributions from bone marrow-derived stem cells [38]. Indeed, several innovative studies in preclinical models of liver diseases highlight the remarkable regenerative capacity of hepatocytes in vivo, pointing out the feasibility of cell therapies for rebuilding failed or diseased liver.
1.2.1 Cell Sources
Different types of cells have been utilized for cell therapies, including primary hepatocytes, immortalized cells, and stem cells. Therefore, several issues should be carefully considered when exploring cell therapies. Ideally, cells should be easy to obtain, non-immunogenic and non-tumorigenic, and stably express the desired functional traits [39].
1.2.1.1 Primary Hepatocytes
Primary hepatocytes are mainly obtained from non-transplantable human liver but also obtained from human fetal liver and healthy animal donors (e.g., porcine liver) [40]. These cells are infused via portal vein and rapidly adhered to existing extracellular matrix to repopulate the diseased liver. Despite showing promise in several studies, the reported amelioration of liver function was modest and not durable [41], largely due to substantial variability in functional activity of isolated primary hepatocytes. Also, these cells are not easily maintained in culture for prolonged times, therefore, alternative cell sources have been sought.
1.2.1.2 Immortalized (Transformed) Hepatocytes
The need for functional hepatocytes with high in vitro and in vivo proliferation capacity led to the development of immortalized human hepatocytes, such as tumor-derived C3A cells (hepatoblastoma subclone) that have been explored clinically with bioartificial liver devices [42, 43]. Immortalized normal hepatocytes have also been utilized for liver disease therapy. Immortalized human fetal hepatocytes showed promising results in mice and demonstrated superior curative metabolic activity in comparison to tumor-derived C3A cells [44].
1.2.1.3 Stem Cells
The therapeutic potential of stem cells is based on their capacity of self-renewal and pluripotent differentiation. The activation of liver stem cells to expand and differentiate for liver regeneration has been explored for various forms of liver diseases, using fetal and adult liver stem cells [37]. However, isolation of these cells remains challenging because they constitute lesser than 1 % of liver mass, making them unfavorable for large-scale preparation [45]. The feasibility for regenerating functional hepatocytes was also evident with non-liver stem cells, such as bone marrow-derived stem cells. Specifically, mesenchymal stem cells have demonstrated remarkable improvement of liver function in several studies [46], besides being multipotent and easily accessible. Induced pluripotent stem (iPS) cells have also been assessed for liver regeneration potential and, indeed, successfully generated functional human liver [47]. In fact, iPS cells are superior to other types of cells in precluding ethical concerns of stem cell technology and potential issues of rejection. Other types of stem cells that have been explored and showed promising potential are human placental cells [48] and embryonic stem cells [49].
1.2.1.4 Xenogeneic Cells
Because of the challenges related to the availability and suitability of human hepatocytes, bovine and porcine primary hepatocytes have also been considered for cell therapy of liver diseases [50]. While maintaining liver metabolic activities, the use of porcine hepatocytes, however, is hampered by their immunogenicity and functional mismatch of porcine proteins with the corresponding human counterparts.
1.2.2 Cell Therapy Strategies for Liver Diseases Treatment
Engineering bioartificial liver substitutes have gained increased interest aiming to develop alternative therapies to liver transplantation. Several designs of cell therapies have been developed such as liver repopulation via hepatocyte transplantation, extracorporeal artificial liver devices, and tissue engineering. Such innovations are currently under intense investigation for future applications in liver disease management.
1.2.2.1 Liver Cell Transplantation
Direct hepatocyte transplantation was proposed as a lesser invasive alternative to whole organ transplantation, especially for severely ill and pediatric patients [41]. Typically, isolated hepatocytes are delivered into the liver through portal or splenic injection. This strategy has been challenged by the modest engraftment efficiency of transplanted cells [51], which limits the applicability of hepatocyte transplantation for inborn metabolic diseases [52] where curative effects are sufficiently driven by minimal recovery of the missing liver function.
1.2.2.2 Extracorporeal Bioactive Liver Perfusion Systems
Attempts to develop artificial liver devices, such as artificial liver support devices (ALSD) and bioartificial liver (BAL) devices, aim to support patients with a liver disease through hemofiltration and detoxification and substituting the missing or insufficient liver function. These devices are often used for short term until the recovery of the transplanted liver or for long term as a chronic supportive therapy. ALSD are non-biological devices similar to renal dialysis units, working through biochemical and biophysical reactions for detoxification when plasma is pumped through cartridges of activated charcoal or immobilized albumin, and hence do not offer metabolic and secretory liver functions [53]. BAL devices are advantageous in providing essential liver functions as they use human C3A or porcine hepatocytes as functional components, which are immobilized on a semipermeable membrane in various configurations such as flat plate or hollow fiber capillaries [54].
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