Molecular therapy for renal diseases

American Journal of Kidney Diseases

The Official Journal

National

of the

Kidney

Foundation

VOL 28, NO 4, OCTOBER 1996

IN-DEPTH REVIEW

Molecular Therapy for Renal Diseases Michael S. Lipkowitz, MD, Mary E. Klotman, MD, Leslie A. Bruggeman, PhD, Paul Nicklin, PhD, Basil Hanss, PhD, Jay Rappaport, PhD, and Paul E. Klotman, MD 0 The introduction of molecular therapy through the delivery of nucleic acids either as oligonucleotides or genetic constructs holds enormous promise for the treatment of renal disease. Significant barriers remain, however, before successful organ-specific molecular therapy can be applied to the kidney. These include the development of methods to target the kidney selectively, the definition of vectors that transduce renal tissue, the identification of appropriate molecular targets, the development of constructs that are regulated and expressed for long periods of time, the demonstration of efficacy in vivo, and the demonstration of safety in humans. As the genetic and pathophysiologic basis of renal disease is clarified, obvious targets for therapy will be defined, for example, polycystin in polycystic kidney disease, human immunodeficiency virus (HIV) type 1 in HIV-associated nephropathy, a-galactosidase A in Fabry’s disease, insulin in diabetic nephropathy, and the “minor” collagen IV chains in Alpott’s syndrome. In addition, several potential mediators of progressive renal disease may be amenable to molecular therapeutic strategies, such as interleukin-6, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-6 (TGF-6). To test the in vivo efficacy of molecular therapy, appropriate animal models for these disease states must be developed, an area that has received too little attention. For the successful delivery of genetic constructs to the kidney, both viral and nonviral vector systems will be required. The kidney has a major advantage over other solid organs since it is accessible by many routes, including intrarenal artery infusion, retrograde delivery through the uroexcretory pathways, and ex vivo during transplantation. To further restrict expression to the kidney, tropic vectors and tissue-specific promoters also must be developed. For the purpose of inhibition of endogenous or exogenous genes, current therapeutic modalities include the delivery of antisense oligodeoxynucleotides or ribozymes. For these approaches to succeed, we must gain a much better understanding of the nature of their transport into the kidney, requirements for specificity, and in vivo mechanisms of action. The danger of a rush to clinical application is that superficial approaches to these issues will likely fail and enthusiasm will be lost for an area that should be one of the most exciting developments in therapeutics in the next decade. 0 1996 by the National Kidney Foundation, Inc. INDEX WORDS: tors; retrovirus

Molecular therapy; vectors; liposomes;

gene therapy; renal disease; antisense oligodeoxynucleotides;

M

adenovirus vectors; ribozymes.

adeno-associated

virus

vec-

OLECULAR therapy through the delivery of nucleic acids either as oligonucleotides or genetic constructs is emerging as a revolutionary and promising form of therapy for the treatment of human disease. For diseases involving the kidney, molecular therapy to replace dysfunctional genes or to suppress the production of disease mediators represents a promising new therapeutic approach. Because it is accessible for

From the Divisions of Nephrology and Infectious Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY; and Drug Discovery, Ciba Pharmaceuticals, Horsham, West Sussex, United Kingdom. Received February 12,1996; accepted in revisedfonn May 23, 1996. Address reprint requests to Michael S. Lipkowitz, MD, Box 1243, Department of Medicine, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. 0 1996 by the National Kidney Foundation, Inc. 0272-6386/96t2804-0@01$3.00/0

American

1996:

Journal

of Kidney

Diseases,

Vol 28, No 4 (October),

pp 475-492

475

LIPKOWITZ

476

direct infusion of therapeutic agents both in vivo and ex vivo during transplantation, the kidney holds tremendous potential for organ-specific therapy. Many barriers remain, however, and many issues must be addressed before molecular therapy will be successful. The major questions to be answered include How can the kidney be targeted selectively? What are the appropriate vectors? What are the best molecular targets? How can gene expression be maximized? How can long-term expression be achieved? and How can we ensure patient safety? This review will address several of these critical questions that must be answered before molecular therapy can be successfully introduced to patients for the treatment of renal diseases. RENAL

DISEASES THAT BY MOLECULAR

CAN BE TARGETED THERAPY

Ideally, the diseases that we would most like to address with molecular therapy are those for which no effective therapy exists, not necessarily those diseases that are most common. Based on the prevalence estimates of renal diseases for the end-stage renal disease (ESRD) population from 1988 to 1991,’ the most common primary diagnoses were diabetes (33.8%), hypertension (28.3%), and glomerulonephritis (12.6%). Cystic disease of the kidney, while accounting for only 3% of ESRD patients, is one of the most prevalent forms of genetic disease worldwide. Advances have now provided the molecular genetic definition of the most common forms of polycystic kidney disease.2,3 Despite this dramatic genetic advance, the molecular pathogenesis of polycystic kidney disease remains unknown and serves as an important area of investigation. Other disorders of lesser prevalence for which a genetic basis has been recently defined and which may be amenable to molecular therapy include Alport’s syndrome,“ Liddle’s syndrome,5x6 and a role for genetic alterations in the angiotensinconverting enzyme gene in progression of renal disease.‘,’ While human immunodeficiency virus-associated nephropathy (HIVAN) accounted for less than 0.3% of ESRD in 1994, a review of New York and Baltimore programs indicates that HIVAN has now surpassed hypertension as a diagnostic category for new patients with ESRD, accounting for 15% of ESRD patients in New York City and 14% of new ESRD patients in

ET AL

Baltimore.’ Thus, with the identification of new molecular targets and the changing demographics of ESRD, potential targets that can be approached with molecular therapy are emerging in our patient population. Replacement Therapy The most straightforward approach to gene therapy is replacement of a dysfunctional gene with a functional copy. This has been attempted for adenosine deaminase deficiency and cystic fibrosis.‘Os’l If the genetic defect in polycystic kidney disease is also a loss of function, this disease may be cured by replacement therapy. It remains to be determined, however, whether this mutation results in a loss of function or a gain in function, and whether the appropriate target for therapy is the large molecular weight gene product that encodes polycystin. Several other replacement strategies that will impact on endstage renal failure include the delivery of a glucose-regulated insulin expression construct in a reservoir tissue for the treatment of diabetes or, similarly, synthesis of a-galactosidase A in Fabry’s disease. Usually, the liver or skeletal muscle are considered reasonable target tissues to serve as reservoirs for production, but the kidney has the advantage of availability ex vivo prior to transplantation and high rates of blood flow. Thus, the kidney may be an ideal site for gene replacement therapy. Inhibition

of Disease Mediators

Multifactorial diseases, while clearly affecting the greatest number of patients, are also the most difficult to address since their pathogenesis is usually the least well understood. Recently, several mediators of renal disease have emerged that may represent common pathways of pathogenesis. Therefore, these mediators may serve as excellent molecular targets. An example of such a target is transforming growth factor-p (TGF-P), which has been implicated in the pathogenesis of extracellular matrix deposition in the interstitium of the kidney and in progressive glomerulosclerosis.12~‘3Enhanced renal expression of TGFfi protein and mRNA has been reported in a range of glomerular diseases, occurring in both animal models’4-19 and in humans.19‘*’ We have detected abundant TGF-/3 within tubulointerstitium in renal biopsy specimens from HIV type 1 (HIV-

MOLECULAR THERAPY FOR RENAL DISEASES

1)-seropositive patients with HIVAN and other renal diseases involving the renal interstitium (unpublished observations). In the animal model of HIVAN, we have found a similar distribution of TGF-P and an increase in mRNA.Z2 These findings suggest that intrarenal production of TGF-P induces renal pathology, in particular the accumulation of extracellular matrix proteins in the interstitium and glomeruli. In vitro, TGF-PI increases production of extracellular matrix proteins by several renal cells types, including glomerular epithelial cells,23 glomerular mesangial cells,24 and tubular epithelial cells,25 and stimulates mesangial cell proliferation, at least in part, by increasing platelet-derived growth factor (PDGF) receptor expression.26 Other factors, such as basic fibroblast growth factor (bFGF), PDGF, and interleukin-6 (IL-6), also have been identified as potential mediators of renal disease and, as such, are also potential targets for molecular therapy. ANIMAL

MODELS

The development of effective molecular therapeutic interventions requires appropriate animal models. Conversely, the absence of such models remains a major barrier to the advancement of gene therapy for all diseases. An ideal animal model for the study of gene therapy in renal disease should have the following characteristics: it should accurately model both metabolically and clinically an important renal disease; it should be small, inexpensive, and readily available; and, critical for gene therapy, it should have conserved targets at the molecular level as the basis for its pathogenesis. One model, the HIV-l transgenic mouse, has many of these characteristics and provides an animal model for exploring the potential of molecular therapy for renal disease. In the murine model, expression of the HIV-l transgene in kidney induces renal disease that is histologically identical to HIVAN in huIn addition, potential targets, such mans. 2’~22827-29 as TGF-/3, that play a role in other diseases are also expressed in HIVAN. This model should allow assessment of the roles of local (intrarenal) versus systemic molecular therapy in ameliorating the development of renal disease, as well as the relative importance of cytokines such as TGF-0, bFGF, and IL-6 in the disease process. Additional animal models of renal disease ex-

477 Table 1. Delivery Systems for Molecular Viral vectors Adenovirus Murine retroviruses Adeno-associated virus Herpesviruses Vaccinia virus Nonviral delivery Cationic liposomes HVJ liposomes Direct infusion of naked DNA Direct injection of naked DNA Microspheres DNA-ligand complexes DNA-polylysine-asialoglycoprotein DNA-transferrin DNA-polylysine-adenovirus membrane

Therapy

complex

ist that are useful for studying human disease, but that at present do not fit the criteria for the study of molecular therapy as well as the HIV1 mouse. For example, mouse models for autosoma1 recessive polycystic kidney disease differ in phenotype from human disease, and chromosomal mapping in the mouse identifies loci distinct from those for human disease.30,3’The nonobese diabetic mouse is a potentially interesting model of autoimmune diabetes mellitus.32 This is a complex multigenic disease linked to at least 14 genetic loci in the nonobese diabetic mouse32 and is, therefore, unlikely to be able to be addressed by molecular therapy to affect the genetic causes of disease. However, it may be a good model for gene replacement therapy using intrarenal delivery of a glucose-regulated insulin gene. GENE

DELIVERY

The success and safety of gene therapy for any disease will depend on (1) the efficient delivery of the exogenous gene, (2) the long-term and appropriately regulated expression of the transduced gene, and (3) the ability to safely target the gene to a particular organ or tissue. Several approaches are presently being explored for delivery of exogenous genes to human cells, including the use of recombinant viral vectors and nonviral systems, such as liposomal delivery or direct DNA injection (Table 1). Characteristics that distinguish available delivery systems include the tropism of the delivery system for vari-

LIPKOWITZ

478 Table

2. Properties

of Viral

Adenovirus

Integration Titer Transduces nonreplicating cells Producer/ packaging lines Clinical experience Immune response Size of inset-l

-

Vectors

Retrovirus

AAV

High

+ +I-

+

-

+

+ + +

+ ++ ~10 kb

-

=lO

kb

+ (site

specific) +/-

? 4.5-5

kb

ous species and cell types, the efficiency of transduction, and the ability of the system to produce either transient episomal expression of the gene or chromosomal integration with long-term expression of the gene. In addition to the characteristics of the delivery system, specifics of the delivered genetic construct, such as the inducibility and specificity of the promoter and the potential toxicity of the expressed gene, are important considerations. Since the majority of diseases leading to renal failure are either genetic diseases or chronic diseases, treatment of these entities will require long-term expression of the therapeutic gene best achieved by integration into the host genome. Site specificity of expression of an exogenous gene in the kidney or in a specific cell type within the kidney can be influenced not only by the choices of the delivery system and the promoter but also by the route of delivery in that the kidney is accessible both in vivo via the renal artery or ureter and ex vivo in the case of transplantation. The accessibility of the kidney, particularly during transplantation, may make it an ideal target organ for molecular therapy. For example, the delivery of an appropriately regulated insulin gene to the kidney ex vivo prior to transplantation into a diabetic patient could be an ideal method of gene delivery. Defining and optimizing the characteristics of delivery systems and defining promoters that are active in specific renal cell types within the kidney are critical areas of ongoing investigation. Although three viral vector systems have been studied extensively (Table 2), only two currently permit integration of a delivered gene: murine

ET AL

retroviruses and adeno-associated virus (AAV). The former system has the benefit of broad tropism, ease of genetic manipulation, efficient and stable integration in dividing cells, a prior history of successful use, and ongoing use in clinical trials for several diseases.33 The requirement for cell division to achieve expression of genes delivered by retroviral vectors is a major drawback to gene delivery in the mature kidney in which only a small fraction of cells are proliferating. Gene Delivery to the Kidney With Retroviral Vectors Two different approaches using retroviral vectors for gene transfer in the kidney have been reported. In one study, retrovirus was directly injected into the renal parenchyma.34 Since retroviruses require cell replication to achieve integration and expression, renal epithelial cell proliferation was induced by tubule injury. Reporter gene expression was detected in 60% of kidneys examined. The major limitation of this strategy is the necessity to induce significant renal injury and the level of renal tissue transduction. A second approach has used retrovirus-mediated transduction of renal cells in vitro followed by reinfusion or implantation of transfected tissue.35”7 In one such study, stably transfected mesangial cells were reinfused into the renal artery, resulting in reporter gene expression in approximately half the glomeruli.37 Induction of native mesangial cell injury and proliferation prior to infusion of transfected mesangial cells resulted in a marked increase in reporter gene expression. While mesangial cell infusion resulted in high levels of glomerular expression, toxicity in the form of an acute reduction in single-kidney glomerular filtration rate (20%) with subsequent expansion of glomerular matrix, increased cellularity, and evidence of sclerosis were detected. In addition, reporter gene expression decreased dramatically by 14 weeks, either from downregulation of the promoter used in these studies or from death of infused cells. Recent studies of retroviral gene delivery in non-human primates raise questions about the utility of current retroviral vector systems. A significant number of animals develop T-cell lymphomas that appear to be related to the presence of an array of replication-competent retroviruses generated by recombination of the thera-

MOLECULAR

THERAPY

FOR

RENAL

DISEASES

peutic construct with helper packaging virus and with endogenous retroviruses in the murine packaging cell line.38 This issue must be resolved if retroviral vectors are to achieve widespread use in humans. Gene Delivery to the Kidney With Adenoviral Vectors An alternative viral delivery technique has been to infuse adenovirus vectors either intraarterially or into the renal pelvis.39 This strategy resulted in significant reporter gene expression in renal proximal tubule cells (intraarterial route) or papillary and medullary tubule epithelial cells (urinary space). Reporter gene expression peaked at 2 weeks, declined significantly at 4 weeks, and was limited to tubule epithelial cells. In a second study, transient transduction of renal cells by adenovirus was limited to endothelial cells; optimal transduction could be shifted from renal cortex to medulla by vasodilator therapy.40 The limitations of adenoviral vectors include the transient nature of gene expression due to lack of integration of the therapeutic gene construct, and the significant immune response to the viral proteins encoded by the current generation of adenoviral vectors. This inflammatory response both limits the effectiveness of adenoviral gene expression4’ and results in inflammation that could result in significant toxicity.42 Presently, efforts are aimed at reducing the immune response by re-engineering the adenoviral vector as well as treatment of the host response.” Gene Delivery to the Kidney With AdenoAssociated Virus Vectors Although not presently used in the clinic, AAV has many attractive characteristics for targeting the kidney. This defective, single-stranded parvovirus is not associated with known pathology in humans or animals despite high levels of silent infection in vivo. The virus is relatively easy to engineer for use as a gene delivery vector. Because it is a replication-defective virus, either adenovirus or herpesvirus is needed to provide helper function and complete an AAV life cycle. This is a useful feature for gene therapy when replication and spread of the virus vector is not desirable.43 Both wild-type and recombinant AAV integrate into the host genome and establish a latent infection efficiently in the absence

479

of the helper virus.44 The wild-type virus has been shown to infect a variety of cell types in vitro and indirectly in vivo. Routine screening shows that 2% of human embryonic kidney cell lots and 20% of African green monkey kidney cell lots are naturally infected, indicating probable in vivo tropism for renal tissue.43 Transduction of a variety of cell lines as well as primary human mesangia145 and other cells46-51has been reported. The wild-type virus efficiently integrates into the host genome, preferentially in a small region of chromosome 19.52,53Such sitespecific integration could eliminate a major concern of gene therapy, insertional mutagenesis. This site specificity appears to be lost in recombinant AAV, but should be recoverable with the ongoing efforts to determine the AAV-specific genes that mediate chromosome 19 integration.54 A unique characteristic of AAV that makes this vector system attractive for renal targeting is the ability of the virus to infect and result in gene expression in nondividing cells, a feature that murine retroviruses do not possess.55*56 While no definitive study has demonstrated that the virus can integrate in quiescent, nondividing cells, it is clear that recombinant virus efficiently transduces and expresses exogenous genes in growth-arrested cells for prolonged periods.55356 Adeno-associated virus delivery of indicator and therapeutic genes directly to the central nervous system of the rat has yielded promising results regarding the transduction of quiescent cells in vivo. Injection of a recombinant virus containing a reporter gene or the human tyrosine hydroxylase gene directly into the caudate nucleus resulted in gene expression, with the latter causing significant behavioral improvement in a rat model of Parkinson’s disease.57 In light of the nondividing state of both brain and renal cells, these results are encouraging. Wild-type AAV has been re-engineered for the production of recombinant virus (Fig 1). Replacement of all the AAV genes with recombinant genes flanked only by the AAV inverted terminal repeat sequences (ITRs) results in successful packaging of the recombinant genes into an infectious virus particle in the presence of a second helper plasmid. The helper plasmid contains all the deleted AAV genes (Cap and Rep) and thus supplies the genetic information for the synthesis of these viral proteins. When both re-

480

LIPKOWITZ Recombinant hived

Piasmid

horn prutQO1

ET AL

Helper Piasmid Derived from pAAWed

AN n-R

Recombinant AAV gene is excised tram psufz?Ol. Rep and adenovirus protehw aid In replication but are not packaged tion: DNA repilcation, RNA transcription, and protein synthesis

Replication-deticient AAV Virions: Cap (viral coat) proteins encase singicstmnded, recombinant AAV DNA

Fig 1. Production scheme for AAV gene delivery vectors. Cultured 293 cells are cotransfected with one plasmid (derived from psub201) containing a therapeutic gene flanked by the AAV Ill&“’ and a second plasmid (pAAV/ad) containing adenovirus ITRs flanking AAV Cap and Rep open reading frames that encode the viral coat and replication proteins.” The AAV ITRs allow packaging of the therapeutic gene into the viral particle, while the genetic material in pAAV/ad cannot be packaged. Since there is no homology between the therapeutic construct derived from psubZO1 and pAAV/ad, homologous recombination generating replication-competent AAV cannot occur. As with wild-type AAV, infection with a helper virus, in this case adenovirus, is necessary for AAV DNA replication, RNA transcription, and protein synthesis. At 40 hours cells are lysed, adenovirus is destroyed by heating the lysate, and replication-deficient AAV particles can be further purified by cesium chloride gradient centrifugation.

combinant and helper plasmids are cotransfected into a producer cell in the presence of a helper virus (adenovirus or herpesvirus), recombinant virus is produced.H*58 The helper plasmid genetic material cannot be packaged into the AAV particle due to the absence of the AAV ITRs, which are essential for packaging. The lack of genetic overlap between the two plasmids prevents the generation of wild-type virus through homologous recombination. The recombinant virus is replication incompetent and, furthermore, recombinant virus could only be produced in vivo if both adenovirus (or herpesvirus) and wild-type AAV simultaneously infected a transduced cell. Recently, the ability to enhance transduction more than loo-fold with recombinant AAV has been reported using gamma irradiation or agents such as cisplatin, hydroxyurea, and topoisomerase inhibitors.593m We have been able to confirm this phenomenon in human mesangial cells.45 Understanding the mechanism(s) involved in this enhanced transduction should lead to novel manipulations of organs either in vivo or ex vivo to improve delivery to nondividing targets.

A potential limitation of AAV as a delivery system is the cumbersome production scheme, particularly in light of the difficulty in establishing stable packaging or production lines (Fig 1). Another potential disadvantage is the limited packaging capability of the virion: the largest therapeutic gene that can be efficiently packaged is 5 kilobases. 61 If additional AAV genes are added back to the viral construct to reconstitute integration site specificity, this may further limit the size of the therapeutic genetic construct. The resulting vectors may be adequate for intracellular expression of antisense sequences or ribozymes but will prohibit the expression of large cellular genes. Nonviral Delivery Systems Nonviral systems, such as liposomes or DNAligand conjugates, also can be designed to deliver genes to the kidney. Gene transfection by cationic 1iposomeP offers several attractive features for gene delivery in vivo. Liposomes are relatively easy to make in large quantity and recent formulations have improved their stability.

MOLECULAR

THERAPY FOR RENAL DISEASES

Theoretically, negatively charged DNA and proteins are complexed by the positively charged liposomes without size or structural limitations. Results of in vitro experiments suggest that liposomes have broad tropism with good transduction efficiency.63 Recent safety studies indicate that liposomes can be effective and nontoxic in vivo.64-68 Localized intrarenal delivery of genes has been accomplished recently using cationic liposomes. Renal artery infusion of DOSPA/DOPE (Lipofectamine; GibcoBRL, Gaithersburg, MD) or DOTMA/DOPE (Lipofectin; GibcoBRL) liposomes resulted in reporter gene expression exclusively outside the vascular compartment in renal cortical and outer medullary tubule epithelial cells in rats69 and mice” with no detectable expression in glomerular cells or inner medullary structures. Infusion of liposomes into the renal pelvis resulted in expression only in cortical and outer medullary epithelial cells, while direct injection into the renal parenchyma produced no expression at all.” A novel variation of liposomal gene delivery has been introduced in the hemagglutinating virus of Japan (HVJ) liposome system. In this system, DNA is incubated with high-mobility group 1 nuclear protein (to facilitate DNA transport into the cell nucleus), introduced into liposomes by sonication, then fused with inactivated hemagglutinating virus of Japan (to improve fusion to cell membranes).“-” Infusion of HVJ liposomes into the renal artery resulted in selective reporter gene expression in glomerular cells (mesangial and/or capillary), with detection in as many as 30% to 40% of glomeruli.72.73 Transduction was transient, with peak levels of reporter gene expression at 4 days. This method, although transducing a minority of glomerular cells, produced glomerulosclerosis when TGF-P or PDGF was introduced into rat kidney7” and ameliorated antiThy l.l-induced acute nephritis by delivery of oligonucleotides.74 There are several issues that need to be further defined regarding intrarenal delivery using liposomes. The transient nature of gene expression using liposomes would require repeated administration. With repeated administration, toxicity will be an important issue that has only been addressed in a system that selectively transfects the intravascular compartment.68’75 In addition,

481

repeated exposure to the protein contained in the HVJ liposomes may generate an immune response that renders further infusions ineffective, as has been reported for adenovirus vectors.42’76 The liposome delivery systems described above were apparently quite selective, as the cationic liposomes resulted in reporter gene activity only in tubule epithelial cells69,70while HVJ liposomes only transfected glomerular cells.69,70The nature of this selectivity remains unclear, but may have resulted from differences in liposome composition,” size,” or intravascular delivery dynamics. Finally, it is possible that the different promoters used are not equally active in all renal cell types and that selective expression of reporter genes is related to promoter strength rather than selective liposomal delivery of the plasmid DNA. Important parameters, such as DNA to liposome ratio, DNA dose, lipid structure, and promoter specificity and strength, need to be compared and optimized for delivery of genes to kidney cells both in vitro and in vivo. This information is critical for understanding the potential of this vector for gene therapy directed to the kidney. As mentioned above, one of the major barriers to liposomal approaches now available is the lack of prolonged expression resulting from the failure to integrate. It may be possible, however, to adapt liposomal delivery for long-term expression and/or integration by incorporating features of the AAV system. Results of our studies as well as those of others using liposomal delivery of a recombinant plasmid containing genes flanked by the AAV ITRs alone in the absence of virus suggest that integration may be achievable with stable gene expression for at least 3 months (preliminary data and refs 79 and SO). Furthermore, transfection of wild-type AAV inserted into a plasmid results in preferential integration on chromosome 19.” These data suggest that long-term expression and perhaps even sitespecific integration can be achieved by incorporating recombinant AAV into plasmids for liposomal delivery to cells. For all methods of gene delivery, the low level of transduction efficiency remains a major unresolved problem that impedes successful clinical application of gene transfer strategies to diseases in humans. Results of recent studies suggest the possibility, however, that effective inhibition of disease progression can occur despite low levels

LIPKOWITZ

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of transduction in vivo. For example, a construct that expressed decor-in was transfected into skeletal muscle using naked DNA, a low-efficiency procedure. Circulating decorin reduced the expression of TGF-01 n-RNA by approximately 30% in renal glomeruli, presumably through interruption of an autocrine-positive feedback loop.‘* Results of other recent studies have demonstrated that cationic liposomes can be used to deliver an oligonucleotide with an NFKB consensus sequence to glomeruli in sufficient quantity to decrease mesangial cell proliferation by 75% in the anti-Thy 1.1 model of glomerulonephritis.83 These studies demonstrate that liposomal delivery can achieve a level of transduction resulting in a change in phenotype. Taken together, these data suggest that levels of transfection obtainable in vivo are sufficient to ameliorate renal disease. Kidney-Specfic

Promoters

There are several unique cellular compartments within the kidney that have been shown to produce potential mediators of disease. For example, bFGF is produced by proximal tubular cells and TGF-/? is produced by both proximal tubular cells and mesangial cells. Targeting these unique cellular compartments may be achievable by careful selection of the gene promoter as well as the delivery system. To date, no ideal renalspecific promoters have been identified. Thus, attempts to restrict expression must initially focus on intrarenal delivery either via the renal artery, via the renal collecting system, or during ex vivo manipulation. Identification of appropriate promoters will further limit the region of gene expression within the kidney. Recently, several promising promoters have been identified that may be useful for targeting renal expression of therapeutic constructs; these are listed in Table 3.84-95As an example, the phosphoenolpyruvate carboxykinase (PEPCK) promoter is expressed in kidney, liver, fat, and intestine, but is most active in the proximal tubule of transgenic mice. Kidney-specific expression has been mapped to a 533 bp region of the PEPCK geneg4xg5and can be enhanced lo-fold (in conjunction with a significant decrease in nonrenal expression) with a 20 bp mutation. Gene delivery by renal artery infusion in vivo, or ex vivo, could limit gene expression to the kidney, and a pro-

Table

3. Mammalian Gene

Gene

Promoters Delivery Size

Human GAPDH Rat fibronectin Mouse y-glutamyl transpeptidase II Mouse y-glutamyl transpeptidase IV PEPCK with P3 (I) mutation Human angiotensinogen NHEl Na/H exchanger Rat epidermal growth factor Canine betaine

For

ST AL

Renal

Expression

1,900 416

bp bp bp

Broad In quiescent cells Proximal tubule

240

bp

Proximal

tubule

533

bp

Proximal

tubule

1,300

bp

Proximal

tubule

1,010

bp

1,010

bp

PT, TAL, DCT, connecting tubule TAL, DCT

507

185 bp

type A Vimentin Human renin

1,600

Rat Kid-l

Untested

878

bp bp

Renal

medulla

Broad/mesangial Juxtaglomerular ASN.l cells Kidney only

cells,

Abbreviations: GAPDH, glyceraldehyde-3 phosphate dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; PT, proximal tubule; TAL, thick ascending limb; DCT, distal convoluted tubule.

moter such as PEPCK could further limit expression predominantly to proximal tubule cells. While the promoters in Table 3 have potential for relatively selective intrarenal gene expression, considerable effort is needed to identify truly kidney and renal cell type-specific promoters to optimize molecular therapy for the kidney. The importance of establishing promoter specificity and efficiency in vivo has been recently demonstrated for hepatic gene therapy. In vivo studies have shown that the human alpha-l-antitrypsin promoter is significantly more effective for intrahepatic gene expression than the PEPCK promoter, while the converse was true in cultured hepatocytes.96-98 THERAPEUTIC

MODALITIES

Many of the potential target genes amenable to therapy are those that are pathologically overexpressed in renal disease. Two modalities of molecular therapy hold promise for reducing expression of these genes: administration of antisense oligonucleotides and ribozymes delivered

MOLECULAR

THERAPY

FOR

RENAL

DISEASES

either systemically or as genetic constructs for intracellular expression. Antisense Oligodeoxynucleotides Therapeutic Agents

as Potential

Antisense oligodeoxynucleotides (ODNs) are short (usually 16 to 30 bases), single-stranded DNA sequences that can inhibit specific gene expression, theoretically, by recognition and binding to a specific nucleic acid target sequence. The primary mechanism of action of antisense ODNs is thought to be through translational arrest by interfering with the RNA-ribosomal complex. Certain modified and unmodified ODNs, however, have been shown to initiate RNAse Hmediated degradation of the RNA target bound by the ODNs. In addition to targeting open reading frames, successful gene inhibition also has been achieved using ODNs that target critical RNA-processing sites, RNA species with critical secondary structure, or sites of protein-nucleic acid interactions. While sequence-dependent effects are the most desirable, important nonspecific effects of oligonucleotides contribute to their biological activity and potential toxicity. This is particularly true of ODNs that have been modified to increase stability, such as phosphorothioates.” Synthetic antisense ODNs have been most extensively explored as molecular therapeutic approaches for the treatment of viral infections and cancer. In vitro, antisense ODNs that target specific viral genes have been shown to inhibit a number of viruses, including hepatitis C virus,‘O” simian immunodeficiency virus,‘o’ Epstein-Barr virus-immortalized B cells,“’ and HIV.102-‘06 Furthermore, antisense ODNs targeting specific oncogenes have been shown to reduce colony formation of several tumor-derived cell lines and to reduce in vivo tumorigenicity. 107-‘og Fewer studies have demonstrated effective antisense ODN inhibition of gene expression and/or disease phenotype in vivo. Studies have indicated that antisense ODNs can result in functionally apparent downregulation of receptors,110-114arneliorate arterial smooth muscle proliferation after angioplasty, ’ l5 extend life expectancy in a mouse model of human leukernia,116 and protect from hepatitis B infection.‘17 Although little toxicity was apparent in these animal models, toxicity to antisense has been described in monkeys at doses of

483

20 pg/g body weight when administered as an intravenous bolus. If antisense is administered more slowly, however, a dose of 80 &g body weight is well tolerated.“* Despite the early success of antisense in vivo, the translation of these efforts to clinical application has been limited by the very short plasma half-life of antisense ODNs, their restricted tissue distribution, and nonspecific effects.” Strategies for prolonging plasma half-life by chemical modification of ODNs have extended their half-life lo- to lOGfold by reducing nuclease-mediated degradation. Some modified ODNs maintain reasonable uptake in cultured cells11g and antisense activity both in vivo and in vitro.120~121One of the most widely applied modifications is the substitution of a thiol group at a nonbridging oxygen position. These oligonucleotides, known as phosphorothioates (S-ODN), are resistant to degradation by exonucleases and retain the ability to induce RNAse H cleavage of the target. Before antisense ODNs can gain broader clinical application, however, a greater understanding of their cellular uptake, metabolism, biodistribution, and toxicity will be required. Following systemic administration of S-ODNs, most recoverable antisense is found in the liver and kidney, with little oligonucleotide detected in other organs examined.122~124Within the liver, S-ODNs are extensively degraded,122’24 but within the kidney, S-ODNs remain intact. Experiments in mice performed in our laboratory demonstrate that SODNs could be detected intact for more than 1 hour after intravenous injection.125 Fractional excretion of S-ODNs was 30% and increased to 50% after infusion of cold competitor, suggesting displacement from a bound source, either in plasma or during reabsorption. In situ autoradiography demonstrated that labeled S-ODNs were, at least in part, filtered and then reabsorbed in the proximal tubule (Fig 2). The molecular basis of antisense uptake into cells has remained one of the critical unresolved issues. Oligonucleotide uptake is a rapid process, reaching steady-state levels within 30 to 60 rninutes. 126~‘27 Loke et a112*demonstrated that oligonucleotide uptake is nucleotide length dependent. Uptake is saturable in a variety of cells’26’27*129~‘32 and is inhibited by coincubation with excess unlabeled oligonucleotide,126~‘28”29”“’ but not by deoxyribose 5’-phosphate, mannose 6-phosphate,

484

LIPKOWITZ

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Fig 2. Renal 3H-oligonucleotide uptake. This autoradiograph depicts the intrarenal distribution of labeled S-ODNs 6 hours after intravenous infusion into a rat. Dark granules indicate that the bulk of S-ODNs localize intracellularly in proximal tubule cells.

or fructose 6-phosphate. lz8 Internalization occurs against a substantial concentration gradient,‘27 is sensitive to decreases in temperature,‘263’289’32decreases 40% to 50% when metabolic energy sources are depleted,lz6 and can be blocked by inhibitors of endocytosis.‘26 These data are consistent with an energy-dependent receptor-mediated process. In addition, a significant portion of uptake appears to occur via receptor-independent, fluid-phase pinocytosis.‘33 Finally, an endocytosis/pinocytosis independent uptake pathway that is upregulated by Ca++ also has been recently described. ‘34 Thus, it appears that ODN uptake is a complex process that involves receptor-dependent endocytosis, pinocytosis, and nonendocytic transmembrane transport processes. Recent studies have implicated DNA-binding proteins in ODN uptake.‘25~‘28~‘30~‘32*‘35 Since proximal tubules rapidly reabsorb S-ODNs through a receptor-dependent process, we have recently used this tissue to identify cell-surface proteins that bind DNA. ‘25Furthermore, we have shown that one such binding protein serves as a gated channel that recognizes and transports ODNS.‘~~ While this finding was somewhat unexpected, such channels for macromolecules have been previously reported for proteins and phage ,,NA.‘37-‘do

Ribozymes as Potential Therapeutic Agents

Ribozymes serve as an additional strategy that may be useful to reduce the levels of target

mRNAs. Ribozymes are RNA molecules with the capacity to bind to target RNA by WatsonCrick base pairing and catalytically cleave the target.‘4’,‘42 RNA catalytic activities have been identified in the RNA component of the tRNA processing enzyme (RNAase P),‘43 group I and group II introns,‘443’45 and the circular RNAs of viroids and virusoids. This latter class of ribozymes comprises a variety of circular RNA plant pathogens and the human hepatitis delta agent. RNA catalytic activity inherent in these circular RNAs is critical for replication of these infectious agents.‘46 For these naturally occurring ribozymes, the minimum catalytic centers have been established experimentally and define three major classes of ribozyme motifs, the “hairpin,” the “hammerhead,” and the “axehead” (delta agent), which have been named according to their predicted two-dimensional structures. The hairpin and hammerhead ribozymes have special utility as therapeutic agents, since the RNA domains responsible for hybridization and catalytic cleavage are completely separable.‘47 Ribozymes, which in nature carry out intramolecular (cis) cleavage, have been modified to recognize intermolecular targets and carry out the trans cleavage of heterologous RNA. Ribozyme cleavage can be targeted to heterologous RNA sequences by altering the nucleotide sequences of the hybridization domain as long as the catalytic cleavage domain is conserved. The hairpin ribozyme can be engineered to cleave target RNAs

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Site TARGET

Bf

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I

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Fig 3. Secondary structure and sequence of three “trans’‘-ribozyme/substrate (target) complexes. Base pairing between ribozyme (dark) and target (light), or within ribozyme or target, is indicated by shaded areas. The site of cleavage is indicated by an arrow. (A) The secondary structure and sequence of the native hairpin ribozyme derived from the negative strand of tobacco ringspot virus is illustrated. N and n represent sequences required for base pairing (hybridization) of the ribozyme and target RNAs. Nucleotide requirements within the catalytic domain have been described extensively by Anderson et al. I” By the appropriate design of sequences comprising helix 1 and 2, the native hairpin ribozyme can be adapted for cleavage of heterologous RNAs containing the sequence bnGUC. (B) Secondary structure and sequence of a hammerhead ribozyme. These ribozymes can be adapted to cleave RNA targets containing the sequence nUX, where n can any nucleotide and X can be A, C, or U. (C) Axehead motif of the hepatitis delta agent ribozyme. RNA catalysis requires the transacting (ribozyme) and targetlcatalytic domains. The target RNA that is cleaved also has sequences required for catalysis.

containing the sequence bnGUC (n can be any base and the preceding base b can be G, C, or U, but not A)‘4’,‘42V’48 (Fig 3A). An extensive compendium of sequences has been tested by Anderson et a1,14’ and the catalytic properties of ribozymes with various sequence alterations have been determined. The sequence requirements for target cleavage by the hammerhead ribozyme are nUX (Fig 3B), where n can be any nucleotide and X = C, U, or A, preferably C. An additional ribozyme motif is found in the hepatitis delta agent.15’ Secondary structural

models, including the pseudoknot’ and axehead, Is2have been proposed for the genomic and antigenomic ribozymes of the hepatitis delta agent. It is possible to separate the hepatitis delta ribozyme-substrate complex into two separate RNA molecules for tram cleavage; unlike the hairpin and hammerhead motifs, however, RNA sequences on the substrate strand are critical for catalytic activity (Fig 3C). Mutational analyses of the substrate strand have enabled prediction of three-dimensional models and determination of sequence requirements for cleavage of the sub-

486

s&ate strand based on the in vitro selection of active variants.153 Based on the nucleic acid sequence of the target and ribozyme catalytic motif of choice, it is relatively straightforward to design hairpin or hammerhead ribozymes to cleave virtually any target sequence in vitro. It may be possible to optimize the length of the sequences involved in base pairing between the ribozyme and target for optimal catalytic cleavage in vitro. Paired regions (helices) that are too long may allow poor product release and, therefore, result in poor reaction turnover (Kcat). Helices that are too short may compromise target recognition (Km). In addition, helix length may affect ribozyme structure, thus altering activity. Finally, it is unclear how important ribozyme turnover is intracellularly; a comparison of the effects of varying helix lengths in vitro and intracellularly is necessary to shed some light on this important issue. A variety of factors may play an important role in determining the efficacy of specific ribozymes as molecular therapy. Multiple cellular proteins may alter the intracellular accessibility of target mRNA to therapeutic ribozymes. Furthermore, RNA transport processes may also determine opportunities for target and ribozyme mRNA to interact, and various promoters and RNA sequence elements may have profound effects on the trafficking and localization of ribozyme and target mRNAs. Provided that ribozyme and target mRNAs colocalize, various RNAbinding proteins and/or unwinding proteins may act to facilitate ribozyme activity. The proteins hnRNP Al and the HIV-l gag nucleocapsid (NCp7) bind RNA nonspecifically and have been demonstrated to increase the activity of certain hammerhead ribozymes.154-‘56 This increase in activity appears to be due to increased efficiency of ribozyme-target binding as well as a helix length-dependent increase in product dissociation.154 Given these effects, ribozyme catalytic function optimized in vitro in reactions containing such “chaperone” proteins may better approximate intracellular catalytic function. Therapeutic ribozymes have been developed that successfully cleave RNA targets in vitro, which may be relevant for renal disease. A number of these ribozymes have been successfully delivered to cultured cells using viral vectors or cationic liposomes. For example, hairpin and

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hammerhead ribozymes have been created that successfully cleave HIV target RNA in vitro and diminish HIV replication in tissue culture systems 157-161; similar ribozymes might be useful for the molecular therapy of HIV-associated nephropathy. Ribozymes targeting the RAS oncogene have demonstrated efficacy in reversing the transformed phenotype in vitro and in vivo.162-164 Mutant ~53’~~ and BCRABL’% oncogenes also have been effectively cleaved by ribozymes in vitro and in cell culture. In addition to cell growth genes, ribozymes engineered to reduce the multidrug resistance (MDR) by targeting the gene MDRl (encoding P glycoprotein) have proven effective in reversing resistance to chemotherapy in tissue culture.167.168Finally, a ribozyme that cleaves IL-6 has been shown to effectively decrease IL-6 expression in cultured human umbilical cells, demonstrating the potential feasibility of using ribozymes against cytokine targets in disease states.16’ In addition to the pathologic states described above, it should be possible to address a broad spectrum of diseases with ribozyme therapy: theoretically, since sites susceptible to ribozyme cleavage are present in virtually all RNA molecules, it may be possible to design ribozyme-based therapies for any disease state in which a reduction in viral and/or host gene expression is desired. SUMMARY

Molecular therapy holds enormous promise for the treatment of a broad spectrum of renal diseases. At present, however, all aspects of this modality are in their formative stages. To best apply therapy, the renal community must continue to carefully define the genetics and pathophysiology of appropriate targets and then to generate pertinent animal models to validate therapeutic approaches. Current viral and nonviral delivery systems must be refined to allow highlevel transduction of specific renal cell types avoiding the generation of an immune response and without toxicity. Tissue-restricted and longterm regulated expression will be difficult to achieve, but are critical in developing successful therapy. Finally, while antisense and ribozymes hold great promise as modulators of gene expression, a better understanding of in vivo mechanisms of transport, specificity, and toxicity of these agents will be required in the future. De-

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