September 14, 1999 - Novel Methods in Gene Therapy for Liver Disease : NIDDK

September 14, 1999 - Novel Methods in Gene Therapy for Liver Disease

Digestive Diseases Interagency Coordinating Committee
National Institutes of Health

Novel Methods in Gene Therapy for Liver Disease

Member attendees

NCI
Jorge Gomez, M.D., Ph.D.

NCCAM
Cheong Chah, Ph.D.

NCRR
Bernard Talbot, M.D., Ph.D.

NIDDK
Frank Hamilton, M.D., M.P.H.
Jay H. Hoofnagle, M.D. (DDICC Chair)
Jose Serrano, M.D., Ph.D.
Rita L. Yeager

NINDS
Robert A. Zalutsky, Ph.D.

NIAAA
Thomas F. Kresina, Ph.D. (DDICC Executive Secretary)
Vishnudutt Purohit, Ph.D.

NNMC
James A. Butler, M.D.

 

Guests from NIH

CIT
Thaddeus Frey

NEI
Runtao He

NHGRI
Wendy Fibison
Richard Hess

NIDDK
Jay Everhart, M.D., M.P.H.
Carol Feld
Mary Beth Kester
Catherine McKeon, Ph.D.
Roland Owens
Sharon Pope

NIAID
Diana Berard

NICHD
Aryan Namboodiri
Karen Winer

NIGMS
Pamela Marino

Other Guests

FDA/CBER
Jong Choi

University of Minnesota Medical Center
Clifford J. Steer, M.D.

Welcome

Jay H. Hoofnagle, M.D., Director of the Division of Digestive Diseases and Nutrition at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and Chairman of the Digestive Diseases Interagency Coordinating Committee (DDICC), welcomed attendees to this DDICC meeting on novel methods in gene therapy for liver disease.

Gene Repair

Clifford Steer, M.D., Professor of Medicine and Cell Biology at the University of Minnesota Medical Center, was the first speaker and presented on the topic of gene repair. He reviewed some of the techniques and systems that have been applied to liver gene therapy, including adeno-associated viruses, lentiviruses, SV40 virus, adenoviruses and liposomes, particularly cationic liposomes.

Dr. Steer next introduced the gene therapy system that he has been working on in his laboratory: the induction of DNA mismatch repair by a chimeric oligonucleotide known as a chimeraplast. The chimeric oligonucleotide is targeted to the asialoglycoprotein receptor that is expressed on hepatocytes. In contrast to the use by many investigators of cationic liposomes, Dr. Steer's system requires the use of anionic liposomes.

The oligonucleotide is chimeric because it is made from both DNA and RNA. For added stability and nuclease resistance, the RNA nucleotides are chemically modified to be 2'-O-methyl RNA. The chimeric oligonucleotide is designed so as to induce a single defined nucleotide change in genomic chromosomal DNA. Once the investigator has identified the target nucleotide he wants to change (adenosine [A], cytidine [C], guanosine [G], or thymidine [T]) and its genomic location, he then designs a custom oligonucleotide that is uniquely and specifically directed against the target. The chimeric oligonucleotide typically consists of a 30-base-pair Watson-Crick double-stranded central region, with the two complementary strands linked by two flanking (T)4 hairpin loops. This 68-nucleotide double-stranded circular nucleotide is linear because its 5' and 3' ends, although situated next to each other in space, are not ligated. The 25-nucleotide double-stranded central region of the chimeric oligonucleotide is nearly identical to a 25-bp stretch of target genomic double-stranded DNA, with a single discrepancy in the middle of the region. There, instead of having the base pair that the genomic region has, the chimera has the base pair that the investigator wants inserted into the genome.

The chimeric oligonucleotides, which were first designed by Dr. Eric B. Kmiec of Thomas Jefferson University, are proposed to hybridize to their complementary genomic targets after nuclear uptake. Because of the chimeraplast's design, there will be a single base mismatch in this oligonucleotide:genomic DNA hybrid, which will be recognized by the cell's DNA repair enzymes and repaired, thus accomplishing the desired change in the genomic DNA.

It is proposed that the oligonucleotid:genomic DNA hybrid forms because of the additional Watson-Crick stability that 2'-O-methyl RNA:DNA base pairs have as compared with DNA:DNA base pairs. The target genomic DNA does not need to be transcriptionally active in the target cell, nor does the target cell need to be undergoing DNA replication. This ability to target noncycling cells, such as are found in the liver, is a significant improvement over other gene therapy techniques that require cell division.

Dr. Steer estimated that up to 100,000 chimeraplast molecules may enter a single cell nucleus and that this molar excess can drive the gene conversion rate to nearly 100% of the target cells. He recounted two modifications in the chimeraplast preparation: the addition of a fluorescent tag to one end of the oligonucleotide to aid in visualization of cellular uptake and disposition and the addition of the polycationic molecule polyethyleneimine (PEI) to the polyanionic oligonucleotide molecule. The PEI addition results in compact 20-nanometer particles that are the optimal size for in vivo uptake by hepatocytes.

Microscopy of HuH-7 liver cells treated with fluorescently tagged oligonucleotides revealed that only about 20% of cells were internalizing the oligonucleotides, which was not enough. Therefore, Dr. Steer and coworkers decided to specifically target the asialoglycoprotein receptor (ASGR) that is expressed on hepatocyte cell surfaces by derivatizing the PEI molecules with the disaccharide lactose, an ASGR ligand. This action markedly improved the transfection efficiency of HuH-7 cells to nearly 100%; however, the PEI-containing constructs proved to be cytotoxic. The researchers then developed an anionic liposome oligonucleotide encapsulation system formulated with the ASGR ligand galactocerebrosides that were noncytotoxic, optimally sized 50-nanometer particles. When added to primary rat hepatocytes, these galactocerebrosides showed high levels of nuclear uptake.

Co-addition of soluble ASGR ligand blocked cellular uptake, thus demonstrating ASGR-mediated uptake of the liposome-encapsulated oligonucleotides. Injection of either PEI or liposome-encapsulated, ASGR-targeted, fluorescently labeled oligonucleotides into the tail veins of laboratory rats resulted in strong hepatocyte uptake, which was inhibited by the ASGR ligand asialofetuin. The fluorescent markers within the rat liver substantially decay over a 24- to 48-hour period.

Dr. Steer mentioned several interesting potential hepatic gene targets for which there are diseases associated with single point mutations: alpha1-antitrypsin, hemophilia A, hemophilia B (factor IX). Because there are no small animal models available for these diseases, Dr. Steer and colleagues decided to introduce a single A to C point mutation (resulting in a serine 365 to arginine 365 conversion) into the factor IX genes of isolated primary rat hepatocytes and into the intact livers of wild-type rats. Colony lift hybridization analysis revealed that up to 20% of treated rat hepatocytes expressed arginine 365 after treatment of hepatocytes with lactosylated PEI-treated chimeric oligonucleotide. Conversion frequencies of up to greater than 50% in rat liver cells were seen upon injection of lactosylated PEI-treated chimeric oligonucleotide into rat tail veins and were accompanied by phenotypic diminution in factor IX activity and a 25% prolongation in activated partial thromboplastin time. Analogous results were seen with hepatocytes from dogs with hemophilia B because of a point mutation in the factor IX gene.

The point mutations and accompanying phenotypic changes introduced into the livers of the wild-type rats were maintained to 72 weeks and were passed on after cell division, as seen in regenerating rat liver following partial hepatectomy.

Dr. Steer discussed Crigler-Najjar syndrome, an inherited deficiency in hepatic uridine diphosphate (UDP)-glucoronosyltransferase (UGT) activity that results in unconjugated hyperbilirubinemia, and its animal model, the Gunn rat. The UGT gene in the Gunn rat contains a single G-deletion that results in a premature stop codon. Dr. Steer and colleagues used chimeric oligonucleotides to correct the gene defect in the Gunn rat by inserting a G-nucleotide into the genome at the sequence AAATGA, forming GAAATGA and thus removing the TGA premature stop codon from the open reading frame. Using the ASGR-targeted chimeric oligonucleotides, these investigators achieved 15-24% insertion frequency in isolated hepatocytes. In vivo treatment of Gunn rats yielded a 20% insertion frequency that was maintained to 1 1/2 years. Western blot analysis demonstrated UGT expression in treated liver homogenates as opposed to no expression in livers from vehicle-treated Gunn rats. Chimeraplast treatment also reduced the serum bilirubin levels in the Gunn rat by greater than 50% over 4 months, with concomitant increases in bile-conjugated bilirubin as detected by high pressure liquid chromatography. Dr. Steer hopes to receive FDA approval soon to begin clinical trials, in collaboration with Dr. Michael Blaese of Kimeragen, Inc., on a group of Amish patients who have a high prevalence of Crigler-Najjar syndrome.

Besides the liver-specific genetic diseases already mentioned, Dr. Steer is also interested in applying chimeraplasty to Wilson's disease, familial hypercholesterolemia, amyloidosis, lysosomal storage disease, phenylketonuria, ornithine transcarbamylase deficiency, and Sly syndrome (mucopolysaccharidosis type VII). He and his colleagues have inserted up to three nucleotides by using chimeric oligonucleotides and are thus in a position to address genetic diseases such as cystic fibrosis delta F508 mutation.

Dr. Steer also discussed a collaboration with Dr. A.J. Lange and D.A. Okar and the strategy of treating non-insulin-dependent diabetes by targeting 6-phosphofructo-1-kinase and lowering the level of hepatocyte glucose output.

Dr. Steer discussed the advantages of chimeraplasty over other gene therapy methods: it does not use viruses; the changes to DNA sequences are performed surgically and precisely and repair the genetic defect; the genomic changes should be identical in all affected cells; it does not involve gene augmentation of additional open reading frames to the genome; it is long lasting, if not permanent; and it is quite simple to perform. He concluded his presentation by thanking all his collaborators and coworkers.

In response to a request from the meeting organizers, Dr. Steer discussed some of the gene therapy initiatives he thought were important:

  • Encourage NIH grant support.
  • Identify new target organs.
  • Identify new target diseases.
  • Develop accurate animal models.
  • Support clinical trials.
  • Optimize chimeric structures and delivery systems.

When asked by an attendee if chimeraplast efficiency could be increased through readministration, Dr. Steer stated that in the Gunn rat model for Crigler-Najjar syndrome, readministration caused the response to go from 25% reduction in bilirubin to 50% reduction. He mentioned that for many genetic abnormalities (e.g., Crigler-Najjar syndrome and hemophilia), 100% response is not required and that a 10-15% efficiency rate of hepatocyte transformation may be sufficient to see real improvement in clinical outcome. Another attendee asked whether there were any sequence requirements for candidate genomic targets. Dr. Steer replied that a GC-rich genomic region seems to work a bit better than an AT-rich region.

In response to a question on applications of chimeric oligonucleotides to bone marrow-targeted gene therapy, Dr. Steer commented that he is working with two University of Minnesota colleagues, Dr. R.P. Hebbel and Dr. C.M. Verfaillie, on gene correction of single point mutations in the beta globin gene in mouse models of sickle cell disease. He cited two challenges: identifying the hematopoietic stem cells and identifying receptors on stem cells that can be used to target and deliver the corrective chimeric oligonucleotides.

An attendee asked Dr. Steer to comment on possible applications of his technology to infectious diseases. Dr. Steer mentioned hepatitis B virus and hepatitis C virus infections as possible targets. An approach might involve the introduction of premature stop codons, but it would involve significant challenges and might require 100% gene conversion of extant viral genomes.

DDDN Liver Gene Therapy Portfolio

Jose Serrano, M.D., Ph.D., Director of Liver & Biliary and Pancreas Programs in the Division of Digestive Diseases and Nutrition of NIDDK, presented an overview of the Division's gene therapy portfolio. Overall, the researchers are interested in basic research in bile formation, liver growth and repair, and gene therapy. They also support basic and applied research in liver transplantation, preservation and storage of liver specimens, and clinical studies on hepatitis and genetic diseases.

Support mechanisms are 80% investigator-initiated R01 grants, with some R29 and R37. Other research areas include tumor biology, liver injury, cholestasis, bile acids, liver regeneration, and gallstones. The budget for gene and cell engineering has increased in the last few years from $1.6 million to $2.2 million and for the Liver & Biliary and Pancreas Programs overall has increased from $26 million in 1996 to $32 million in 1999.

Specific interests in gene and cell engineering include hepatocyte engineering, gene transfer methodologies, optimization of vectors, and mechanism of action studies.

Trans-NIDDK Gene Therapy Program

Catherine McKeon, Ph.D., Senior Advisor for Genetic Research in the Division of Diabetes, Endocrinology, and Metabolic Diseases of NIDDK, discussed the history and current status of extramural support from NIDDK for gene therapy. In the late 1980s, NIDDK decided to support basic development of gene therapy and issued a Request for Applications (RFA) in basic techniques of gene therapy for genetic diseases. Currently, NIDDK funds $13 million in extramural gene therapy research.

Unlike other parts of the NIDDK research portfolio, the gene therapy program is a little unusual in that it does not rely mainly on research grants. The program also uses pilot and feasibility grants, core centers, and small business innovation research (SBIR) grants. The SBIR grants are important because many small businesses are involved in gene therapy.

Initially, NIDDK used RFAs for gene therapy to generate a sufficient submission rate from investigators. Now, gene therapy is no longer so new and experimental. As a result, a sufficient number of proposals are being submitted, and the RFAs are not needed. Experimental and high-risk gene therapy protocols, however, are best funded through pilot and feasibility grants and have been funded since 1997. The current program announcement (PA) is PA-99-036 for R21 grants for $100,000 maximum for up to 2 years. Approximately 10 per year are being funded. The goal is to develop novel vectors and delivery systems targeted against inborn genetic diseases, for when a sustained, life-long therapeutic effect is required. Examples are long-term expression systems and repeat administration protocols.

Dr. McKeon next discussed the NIDDK gene therapy core center program, a P30 award in existence for about 5 years. These awards are for university-based facilities providing technological and resource assistance to gene therapy investigators campuswide. Examples include vector cores, animal cores, and histology cores. Four gene therapy core centers are currently funded by NIDDK.

NIDDK also participates with other NIH Institutes in supporting the National Gene Vector Laboratories, which manufacture clinical-grade gene therapy vectors, undertake toxicity studies, and support clinical trials in gene therapy. Dr. McKeon noted that they have initiated a master file on adeno-associated virus (AAV), which is currently in clinical trials for hemophilia and is being considered for several liver gene therapy projects. This AAV master file contains toxicological and manufacturing (cGMP) data on AAV vectors, and other clinical investigators can refer to this AAV master file when filing with the FDA, rather than repeating the AAV toxicology studies themselves.

NIDDK Strategic Plan

Ms. Carol Feld, Associate Director for Scientific Program and Policy Analysis at NIDDK, discussed the implementation process for the NIDDK Strategic Plan. The recommendation that NIDDK, along with all Institutes within the NIH, promulgate a Strategic Plan came out of a congressionally directed Institute of Medicine (IOM) study on improving NIH's setting of priorities and public input. Among the IOM recommendations that have been put in motion are that all NIH Institutes establish an Office of Public Liaison, that a Council of Public Representatives to the NIH Office of the Director be established, and that the Strategic Plans be formulated (they are due to be submitted in draft form to the NIH Director by December 31, 1999).

The NIDDK Strategic Plan will have the following features:

  • Organization by cross-cutting scientific themes, rather than by diseases within the NIDDK research mission.
  • A catalog of scientific opportunities and needs.
  • Implementation strategies (what tools, reagents, techniques, and personnel are needed).
  • Five-year time horizon.
  • Public involvement.
  • A final document written in lay language.
  • A widely distributed plan, both electronically and in hard copy.

Requests for public input have been made to more than 70 voluntary and professional organizations with interests within NIDDK's disease mission, and a request for public input has also been posted on the NIDDK Web site. In addition, all of the Interagency Coordinating Committees (e.g., DDICC) have been enlisted to supply input into the NIDDK Strategic Plan.

The NIDDK Strategic Plan, which has also been the topic at several of the recent NIDDK Advisory Council meetings, will include sections on the following topics:

  • Background--Magnitude of the burden of illness.
  • Ongoing NIDDK planning processes.
  • Highlights of the NIDDK research programs and accomplishments.
  • Institute goals and objectives.

Five cross-cutting working groups have been established, each one chaired by a senior member of the NIDDK scientific staff:

  • Genes and Disease--Regulation, Expression, Screening.
  • Cells--Integration of Biological Mechanisms in Health.
  • Causes and Mechanisms of Disease and Injury.
  • Prevention and Treatment of Disease--Epidemiologic and Clinical Investigation.
  • Infrastructure--Human Resources, Technology, and Research Facilities.


Congressional Report

Ms. Rita Yeager, Senior Program Analyst in the Office of Scientific Program and Policy Analysis at NIDDK, presented an update on the status of the Department of Health and Human Services Appropriations Bill for fiscal year 2000 that is before the U.S. Congress. Ms. Yeager reported that this bill was still being marked up in the House and Senate subcommittees. House members were proposing an increase in the NIH budget of $1.35 billion; Senate members were proposing an increase of $2 billion for NIH; and the White House was proposing an increase of $0.32 billion.

Conclusion and Adjournment

Dr. Hoofnagle called on representatives of the Institutes present to provide an update on initiatives involving digestive diseases or gene therapy. He talked about a recent RFA that NIDDK had issued together with other Institutes and had contributed $3 million. Dr. Jorge Gomez of the NCI spoke about the NCI P50 center grants that have included digestive diseases, such as liver cancer and gastrointestinal cancers. Dr. Hoofnagle drew the attendees' attention to the 10th International Symposium on Viral Hepatitis and Liver Disease that is being sponsored by the Centers for Disease Control and Prevention and to an RFA on food safety and food-borne illness that NIDDK is drafting.

Dr. Hoofnagle noted that the next DDICC meeting is scheduled for December 14, 1999. The meeting adjourned at 10:30 a.m. on September 14, 1999.


Page last updated: September 24, 2007

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