RIKEN　Discovery Research Institute
Molecular Cell Science Laboratory
Chief Scientist, Dr. Hiroshi Amanuma

Retroviruses infect living cells, integrating their own genetic information into the DNA of the host cell, and replicate. Through utilization of their replication mechanisms, retroviruses are used as "vectors," i.e., vehicles to deliver genes into cells, in basic research and in clinical applications such as gene therapy. "We are interested in adding new, value-added 'targeting' to retroviral vectors. To that end, we have been developing retroviral vectors that enable cell-specific gene transfer," explained Chief Scientist Dr. Hiroshi Amanuma. The focus of Dr. Amanuma's research has been on "direct targeting." The idea was originally conceived as a promising targeting strategy, and many researchers had been working on its implementation, but there were no reports of unequivocal successes for an extended period of time. It was this environment into which the Molecular Cell Science Laboratory presented the first clear proof that targeting of retroviral vectors can be accomplished through direct targeting.

Retroviruses integrate genetic information into the DNA of host cells

Retroviruses were discovered in 1908 in the form of a tumor virus that causes leukemia in chickens, and have since been found in a wide range of animals, including fish, reptiles, and mammals, as well as birds. Retroviruses are round particles with a diameter of 100 nm (1/10000 mm) (Figure 1). They consist of an outer envelope from which envelope proteins protrude and an inner core containing two copies of a single-stranded RNA that carry their genetic information. "Retroviruses infect living cells and then replicate within the infected cells. The idea of using retroviruses as vectors, in other words as vehicles to deliver genes into cells, developed out of studies of their replication mechanisms," explained Dr. Amanuma.
The envelope proteins of retroviruses bind to membrane proteins located on the surface of cells called "viral receptors" (Figure 2). Once an envelope protein has bound to its appropriate viral receptor, the envelope fuses with the cell membrane and the viral core enters the cell. Immediately after entry, double-stranded viral DNA is synthesized by the reverse transcriptase present in the core using the single-stranded viral RNA as a template. This viral DNA enters the nucleus of the cell and is integrated into the DNA of the host cell as a provirus^*1. Large amounts of viral RNA and viral proteins are produced based on this proviral DNA and assembled at the cell membrane; they then are covered by the cell membrane and bud from the cell as progeny viruses. The progeny viruses then infect other cells, and the cycle begins again; meanwhile, the proviruses are passed from one generation to the next at each cell division.
"The use of retroviruses as vectors would be problematic if the viruses replicate or if their oncogenes are expressed and promote oncogenesis in the cells. So usually the genes required for their replication and oncogenes are removed, and instead a gene that is to be introduced into the cells is inserted into the retroviral genome. Only the ability to transfer genes into cells is left intact in a retroviral vector. Transfection of a retroviral vector DNA into the packaging cells^*2 gives us a retroviral vector which introduces genes into the target cells efficiently (Figure 3)."
Retroviral vectors have applications in basic research as well as gene therapy. For example, in basic research, the functions of a specific gene can be studied by observing the changes that occur when the gene is introduced into cells by retroviral vectors. On the other hand, retroviral vectors are used in gene therapy to treat disease by introducing corrective genes for a particular disease into cells.

Problems with retroviral vectors

Since the first example in 1990, gene therapy clinical trials have been performed on nearly 4000 patients worldwide. "There are still many issues with gene therapy, however, and there is much room for improvement of the vectors," explained Dr. Amanuma, in reference to the current state of gene therapy.
Today, gene therapy is performed on humans using one of two principal approaches: the ex vivo method, or the method of direct administration to the affected area of the body. Ex vivo therapy involves the introduction of a corrective gene into cells obtained from a patient and returning the cells to the patient's body following culturing. "Ex vivo gene therapy is effective on certain types of cells, such as hematopoietic cells, but it has limitations when used on cells of organs. Gene therapy's future lies in part in the development of in vivo methods." In vivo therapy involves the direct administration of vectors into a patient's body, as when a patient takes oral medications or receives an injection of drugs. However, there is one critical problem with in vivo therapy.
Our bodies are made up from 60 trillion cells of hundreds of different types. The difficulty with in vivo therapy lies in the fact that, while carrying genes into the target cells, retroviral vectors also transfer genes to a variety of other types of cells undergoing cell division.
"The infectivity of retroviruses depends on the presence of viral receptors. However, for animals, retroviruses are foreign entities that cause disease. It is unlikely that cells produce these viral receptors solely for the purpose of helping retroviruses to cause infection."
The genes encoding viral receptors have been isolated and sequenced, and the physiological functions of these receptors have been revealed within the last 10 years. mCAT1 is one of the receptors for mouse leukemia viruses (MLVs), which are the most commonly used retroviral vectors today. The primary function of mCAT1 is membrane transport; i.e., the transport of amino acids into cells across the cell membrane. Since membrane transport is a fundamental, indispensable function of living cells, mCAT1 is expressed in most types of cells. This provides an explanation as to how MLVs are able to infect a variety of cell types. "It appears that MLVs have evolved to gain this ability by adapting their envelope proteins to match the mCAT1."
There are several types of MLVs. The MLVs that use mCAT1 as their receptors are categorized as ecotropic viruses, which only infect mouse and rat cells. The MLVs used in gene therapy are categorized as amphotropic viruses, which can infect both mouse and human cells, and use PiT2 as a viral receptor. Since PiT2, which is involved in the membrane transport of inorganic phosphate, is expressed in many different types of cells as with mCAT1, the use of viruses of this type as vectors do not allow the selective introduction of corrective genes to specific cells. That being the case, not only is a high therapeutic effect unlikely, but there exists the potential for adverse reactions caused by gene transfer to other cells. " 'Targeting', which gives us the ability to transfer corrective genes selectively to the target cells, is essential for safer and more effective gene therapy." Vectors can be broadly classified into two categories: vectors derived from viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses) and vectors not derived from viruses. However, targeting is a common challenge for all vectors.

The challenge of targeting

"The first report of a study demonstrating the feasibility of retroviral vector targeting was published in 1994."
The concept of "direct targeting" involves redirection of the cell-tropism of the vector by modifying the specificity of the envelope protein in binding to the receptor, so that a different membrane protein may function as a viral receptor.
Among the variety of membrane proteins that are present on the surface of cells, certain proteins, such as mCAT1 and PiT2, are expressed in many types of cells, whereas other membrane proteins are expressed only in specific cell types.
If the vector is redirected to the latter kind of membrane proteins, then this vector becomes a targeting vector to the specific types of cells.
Many researchers began actively pursuing studies on the direct targeting; however, the success rate was quite low, and the negative reports gradually began to accumulate. "It was our belief that direct targeting is a technique that is simple yet has great potential for future development. We needed to see that it was truly hopeless with our own eyes, and so we began exploring the possibilities."
The relationship between a viral receptor and a retrovirus envelope protein can be likened to that of a lock and key. In order for an envelope protein to bind to a membrane protein other than its normal viral receptor, the shape of the "key," i.e., the envelope protein, must be altered so that it fits the new "lock." The questions were, to what should the shape be changed, and how? Dr. Amanuma's search had begun.

Success in direct targeting

The first candidate selected by Dr. Amanuma to be used for modifying the key was human epidermal growth factor (EGF), one of the molecules involved in cell proliferation. EGF binds specifically to a membrane protein called EGF receptor, which is located on the cell surface. Creation of a chimeric (fusion) envelope protein by genetically inserting the amino acid sequences of EGF into the amino acid sequences of an envelope protein will in theory cause the protein to gain the ability to bind to EGF receptors in addition to the original viral receptors. This may lead to the realization of gene transfer selectively to cells expressing EGF receptors.
"However, we had little information about the proper insertion site for the amino acid sequences of EGF. Fortunately, the three-dimensional structure of the envelope protein of one ecotropic MLV was partially elucidated in 1997, while we were going through a process of trial and error with the related virus. This turned out to be extremely helpful (Figure 4)."

Dr. Amanuma focused on variable region A (VRA) of the envelope protein, which is the site of key importance in the binding of the protein to its natural viral receptor. He selected six random sites (sites 1 to 6) within the VRA and inserted EGF (a ligand ^*3), which consists of 53 amino acids. This experiment revealed that retroviruses with EGF chimeric envelope proteins were produced only when the amino acid sequences were inserted into site 3; insertion into other sites did not result in incorporation of the chimeric envelope proteins into viruses.
Dr. Amanuma tried the similar type of experiment with human stromal-derived factor-1 (SDF-1) as a ligand, a chemokine that plays a role in immunity, consists of 68 amino acids, and binds specifically to the receptor membrane protein CXCR4. He obtained the same results as with EGF. "The use of site 3 allows the production of retroviruses possessing chimeric envelope proteins regardless of the amino acid sequences inserted. In other words, we discovered a universal site that can be utilized to induce alterations in envelope proteins." Other research groups in the U.S. and Canada contemporaneously and independently discovered the same site 3 using different "ligands."
The next question lay in whether the altered retroviral vectors would actually use the intended membrane protein as a viral receptor. "The major difficulty that had been seen in previous studies was that the vectors could bind to the targeted membrane proteins but the envelopes would not fuse with the cell membranes."
But the results of his experiments were even better than anticipated. Not only did the retroviral vector altered with SDF-1 bind to CXCR4, its envelope also fused with the cell membrane and the gene transfer occurred, meaning that CXCR4 actually functioned as a new receptor for MLV. "By inserting the sequences of SDF-1 into an envelope protein, we succeeded in expanding the cell-tropism of the retroviral vector derived from the ecotropic MLV. For human cells, this means transfer into cells expressing CXCR4 but not into cells not expressing CXCR4 (Figure 5)." This was the first clear demonstration that retroviral vector targeting was possible through direct targeting.
Improving gene transfer efficiency remains a challenge for the future. While the retroviral vector altered with SDF-1 allows gene transfer via CXCR4 as well as by mCAT1, which is the original receptor for ecotropic MLVs, the transfer efficiency dropped to below 1/100 with CXCR4 compared with gene transfer via mCAT1. "We have already begun research from several different approaches into ways to achieve a hundredfold increase in efficiency."

Tools for basic research and gene therapy

Although retroviral vectors with envelope proteins altered by the insertion of EGF at site 3 bound to EGF receptors, membrane fusion failed to occur. What was the reason behind these divergent results? "It may well be that not just any membrane protein can serve as a receptor for MLVs." speculated Dr. Amanuma. Membrane proteins can be broadly grouped into two categories: single-pass transmembrane proteins, which pass through the lipid bilayer once, and multipass transmembrane proteins, which pass through the lipid bilayer multiple times. The natural receptors for MLVs, including mCAT1 and PiT2, are all multipass transmembrane proteins, while EGF receptor is a single-pass transmembrane protein and CXCR4 is a multipass transmembrane protein. "Thus far, we have only tested one of each type. Obviously, we need to test more membrane proteins in future studies, but it is possible that only multipass transmembrane proteins can serve as receptors for MLVs."
"The major accomplishment of this work was that we were able to provide two guidelines for future research into direct targeting; those being that the candidates for new viral receptors for MLVs should be multipass transmembrane proteins, and that site 3 should be used to insert amino acid sequences for the purpose of creating chimeric envelope proteins," stated Dr. Amanuma. This research was reported in the September issue of "EMBO reports", published by the European Molecular Biology Organization.
"Our primary objective is to provide tools with broad applicability in various fields such as basic research and gene therapy." The Molecular Cell Science Laboratory is also studying the mechanisms and regulation of a number of cellular functions, such as cell proliferation, differentiation, and histogenesis. "Cell-specific delivery targeting provides a powerful tool for this type of basic research as well, and there is no question that it is useful in gene therapy, although it needs to be implemented carefully while considering the possible risks associated with retroviral gene transfer." The applicability of gene therapy has been broadened to include such diseases as cancer and AIDS, and now the targets of gene therapy are not limited to genetically inherited diseases. Someday, many diseases may become targets for gene therapy.
The need for cell-specific gene transfer will continue to increase in the future. The findings obtained at the Molecular Cell Science Laboratory hold great promise to encourage the related studies based on the old concept "direct targeting".

RIKEN Brain Science Institute
Developmental Brain Science Group
Laboratory for Developmental Gene Regulation
Laboratory Head, Dr. Hitoshi Okamoto

The human brain controls a variety of functions, such as cognition, movement, learning and memory, and emotion through an enormous network consisting of tens of trillions of precisely interconnected neurons. "There has to be a set of rules inscribed in the genome that governs the development of the brain. Our ultimate goal is to find out how the program for development of the brain is executed once it is loaded from the genome, causing each region of the brain and the nervous system to develop into a neural network, and how it enables the brain's functions," stated Laboratory Head, Dr. Hitoshi Okamoto. In the past, developmental biology studies were predominantly conducted in Drosophila (fruit fly) and Caenorhabditis elegans (nematode). The Laboratory for Developmental Gene Regulation is attempting to track down all of the gene clusters involved in the development of the vertebrate brain, through the use of zebrafish. The results will provide invaluable information for many disciplines, particularly in the field of regenerative medicine.

Universal principles of development applicable across species

Homeobox genes were discovered in the 1980s through studies of mutations in Drosophila. Homeobox genes control the development of both the anteroposterior and dorsoventral axes of the body, and determine the morphological characteristics of each body segment. For example, a fly with a mutation in a homeobox gene may develop with legs growing out of its head, or may develop with extra wings.
Later studies involving comparisons of genes from various plants and animals revealed, surprisingly, that all multicellular organisms have homeobox genes similar to those of Drosophila, suggesting that the molecules and basic principles found in Drosophila are also involved in the developmental processes of the body segments and brains of vertebrates, including human beings.
"The discovery of homeobox genes that regulate development across species was the number one hit in developmental biology during the 1980s and 1990s. However, this discovery also begs the question of why are we humans and not flies." If the "directors," in other words the genes that provide the essential directions for development, are fundamentally the same, then there must be differences between the genes of humans and the genes of flies that come into play at lower "stages." Many of the genes involved in the development of Drosophila have been discovered through the creation of thousands of mutant embryos.
"Large-scale mutation studies comparable to those conducted in Drosophila need to be performed in vertebrates as well. We are interested in searching for answers to the question of what made us vertebrates through studies of the novel genes that will be discovered in these experiments."

Studying vertebrate neural differentiation in zebrafish

Hunting for mutations in the nervous system by sifting through 600,000 eggs

One method of searching for the genes involved in neural differentiation is to employ mutagenesis to create a large number of mutant embryos; in essence, this method involves causing mutations in all genes, finding organisms with nervous system abnormalities, and then identifying the causative genes for a particular abnormality.
Treating male zebrafish with a certain agent causes random mutations to occur in their sperm. The progenies resulting from matings between these males and normal females (F1) will have mutations in 20 to 30 genes each. Subsequently, the progenies resulting from matings between F1 males and females of different lineages (F2) will have an average of 50 mutations each. Matings between animals from the same F2 family (siblings) will result in a portion of the progenies inheriting the same mutations as their parents. Thus, different mutations will be seen for each family.
If zebrafish have 40,000 genes and each F2 family has 50 mutations, it can be assumed that mutations will be caused in every gene if it is possible to produce 800 F2 families (or 1500 F2 families if duplicate mutations are taken into account). If eight pairs from each of the 1500 families are mated and approximately 50 eggs are laid for each pair, the result is 600,000 eggs. "We observe the development of each and every one of those 600,000 eggs as we search for nervous system abnormalities."
The process of "sifting" to determine the presence of abnormalities based on a certain criterion is called "screening." At present, large-scale mutation screening - "large-scale" meaning to target all the genes of a vertebrate species - is being solely conducted in the zebrafish and the medaka. Screening in the zebrafish is being conducted by Dr. Okamoto's group only in Japan and by groups in the U.S. and Germany; screening in the medaka is being conducted in Japan and Germany.
Large-scale screening requires that a foundation based on an accumulation of studies and technology be in place, as well as the presence of highly competent personnel. "There might be a once-in-a-lifetime mutation that appears only in one out of 600,000 eggs. The quality and motivation of the people who perform the screening are of key importance. We do have students perform screening as well, but only after they complete an intensive two-week training period. We begin with a basic explanation of what genes are and move through the mechanisms of the nervous system, how we are able to find causative genes by studying mutations, and the techniques involved. We allow them to perform screening once they have gained a complete grasp of the concepts of the study. It is important to provide them with encouragement and to keep their motivation at a high level, so that they can find the mutations that have significant meaning for neural development."

Following the trail of glowing neurons to mutations in the zebrafish

The search for gene clusters downstream of specific genes

Neurons exert specific functions by forming connections to specific sensory organs or muscle cells. What sort of mechanisms enable precise interconnections to form within the brain and nervous system remains an enigma. Both substances that attract neurons (e.g., netrin) and substances that repel neurons have been found in the regions of the spinal cord where nerves intersect. However, their presence alone is not sufficient to explain the mechanisms behind the precise interconnections. "For example, even if the pattern of motor neurons is reversed experimentally, certain motor neurons always manage to connect to specific muscles. The fate of motor neurons, and by that I mean which motor neurons connect to which muscles, is determined as soon as each neuron gains its own identity."
Figure 6 is a schematic illustration of the spinal cord neurons of zebrafish. At around 24 hours after fertilization, each of the primary motor neurons, called RoP, MiP, and CaP, has extended to a different location. Dr. Okamoto's group discovered that, prior to 16 hours postfertilization, i.e., when the identity of each motor neuron is determined, a molecule called Islet-1 and a similar molecule called Islet-2 are expressed specifically in RoP and CaP, respectively (Figure 7). For example, if the expression of Islet-2 in CaP is suppressed, neurons will extend in a direction different from the original direction.
Islet-1 and Islet-2 are LIM homeodomain transcription factors. Transcription factors are molecules that act as on/off switches for genes. While the homeobox genes control regional patterning, LIM homeodomain transcription factors are expressed only in certain cells.
"Since different types of LIM homeodomain transcription factors bind and form pairs, producing many different combinations, these transcription factors are suitable for use in determining the identities of individual cells."
It is suspected that Islet switches on genes that are involved in the expression of proteins that are necessary for the development of the identities of individual neurons; however, the actual target genes have not been identified.
The research team is investigating genes whose expression is suppressed in response to suppression of Islet, currently focusing on genes associated with Islet-3 that are expressed in the midbrain and the midbrain-hindbrain boundary. The midbrain develops into the optic tectum, which controls vision, and the midbrain-hindbrain boundary develops into the cerebellum, which controls movement.
"Experiments performed on chickens have revealed that the optic tectum and cerebellum do not differentiate independently of one another, but that the regions that develop into the cerebellum and optic tectum differentiate while mutually providing molecules to and interacting with one another. This phenomenon is known as reciprocal inductive interaction. For example, in chickens, experimental transplantation of cerebellum into the anterior region of the brain causes the surrounding area to develop into the optic tectum. Islet-3 is suspected to be one of the molecules that regulate the level of reciprocal inductive interactions. When expression of Islet-3 in the midbrain is suppressed, the midbrain will still become the primitive optic tectum, but the cerebellum will not be formed."
The research team has already found roughly 30 genes whose expression is suppressed in response to the suppression of Islet-3, including novel genes with unknown functions and genes already known to be involved in the inductive interactions, such as FGF8 and Wnt1. FGF8 and Wnt1 are secreted proteins that promote cell differentiation and growth.
"Not only does the brain develop though inductive interactions, but the arms and legs do as well. Interestingly, it is known that certain molecules, such as FGF8 and Wnt1, that are involved in the brain's development are also involved in the development of the arms and legs. The brain, arms, and legs are examples of organs that have undergone dramatic changes during the process of evolution."
Inductive interactions may be an important key concept for understanding evolutionary diversity.
"For example, once evolution reached the level of mammals, the telencephalon increased in size and the cerebral cortex developed. It has been revealed that inductive interactions that take place between the outermost tip of the brain and the exterior portions of the brain are involved in the formation of the cerebral cortex. A recent report published in the July 19, 2002 issue of the U.S. scientific journal "Science" described a study conducted in mice which demonstrated that activation in the brain of a substance called -catenin, which is known to interact with Wnt1, dramatically increased the size of the cerebral cortex, to the point where it could not fit in the cranium, resulting in the formation of folds."
The human cerebral cortex has folds, but the cerebral cortex of a normal mouse does not. Does this suggest the possibility that the molecules involved in inductive interactions played an important role in the evolution of the human brain?
"Our immediate and pressing goal is to determine all of the molecules acting in roles on the 'stage' of neural differentiation in zebrafish through a variety of methods, which include searching for the genes that cause mutations and searching for gene clusters that play roles downstream of specific genes, such as Islet. I expect that we can come closer to some of the mysteries of species diversity and evolution of the brain by comparing the functions of those genes in zebrafish with their functions in other animals, such as humans and mice."

Providing essential knowledge for regenerative medicine

Once elucidated, developmental mechanisms will become indispensable knowledge for the field of "regenerative medicine," the aim of which is to regenerate organs that have lost their functionality through the culturing of a patient's own cells.
"As an illustration of this point, the mechanisms behind the differentiation of motor neurons will be useful for the culturing of motor neurons. However, even if culturing motor neurons is possible, for example, a finger may end up moving in the opposite direction, unless the neurons are made to form connections to the proper muscle. In order to achieve the correct connections, we need to know the mechanisms behind those connections as well.
"Now that we have been freed from the prodigious task of sequencing genes, which in the past had consumed a significant amount of time and labor, we can focus our energies on studying the functions of genes involved in development, which is the most interesting theme in developmental biology. Finally, the day has arrived when developmental biology is ready to contribute to mankind by providing valuable data for the field of medicine."
Developmental biology is entering a new stage of development, ushered in by studies in zebrafish.