Invented by Phillip A. Patten, Volker Heinrichs, Willem P. C. Stemmer, Codexis Mayflower Holdings LLC
The market for RIMNA is still in its infancy, but it is expected to grow rapidly in the coming years. The global market for gene editing technologies is projected to reach $10.6 billion by 2025, with a compound annual growth rate of 18.2%. RIMNA is expected to be a major contributor to this growth, as it offers several advantages over other gene editing technologies.
One of the key advantages of RIMNA is its precision. Unlike other gene editing technologies, RIMNA allows for the precise insertion of DNA sequences into specific locations in the genome. This precision reduces the risk of off-target effects, which can cause unintended changes to the genome and lead to unwanted side effects.
Another advantage of RIMNA is its versatility. RIMNA can be used to insert DNA sequences of any length, which allows for the creation of complex genetic modifications. This versatility makes RIMNA ideal for a wide range of applications, from correcting genetic defects to developing new therapies for diseases.
The market for RIMNA is expected to be driven by several factors, including the increasing demand for gene therapies, the growing prevalence of genetic diseases, and the increasing availability of funding for genetic research. In addition, the development of new RIMNA technologies and the expansion of the RIMNA product pipeline are expected to further drive growth in the market.
Despite its potential, there are still several challenges that need to be overcome before RIMNA can become a widely adopted technology. One of the biggest challenges is the development of safe and effective delivery methods for RIMNA. Currently, most RIMNA delivery methods rely on viral vectors, which can cause immune responses and other side effects.
Another challenge is the regulatory environment. As with any new technology, RIMNA will need to undergo rigorous testing and regulatory approval before it can be used in humans. This process can be time-consuming and expensive, which may limit the adoption of RIMNA in the short term.
In conclusion, the market for RIMNA is expected to grow rapidly in the coming years, driven by the increasing demand for gene therapies and the growing prevalence of genetic diseases. While there are still several challenges that need to be overcome, the potential benefits of RIMNA make it a promising technology for the future of genetic engineering.
The Codexis Mayflower Holdings LLC invention works as follows
The article provides methods for modulating, tweaking, improving, and evolving the splicing of proteins and RNAs.
Background for Recombination with insertion-modified nucleic acid
Nucleic acid shuffling allows for rapid evolution in both vitro and vivo of nucleic Acids. Rapid evolution allows for the production of encoded molecule (e.g. nucleic acid and proteins) that have new or improved properties. Through shuffling, proteins and nucleic acid of industrial, agricultural, and therapeutic value can either be created or enhanced. The inventors’ and their coworkers’ publications describe the technology of nucleic acids shuffling. For example, Stemmer et al. (1994) ‘Rapid Evolution of a Protein’ Nature 370:389-391. Stemmer (1994),?DNA shuffling by random fragmentation and reassembly: In vitro recombination for Molecular Evolution? Proc. Natl. Acad. USA 91:10747-10751; Stemmer U.S. Pat. No. 5,603,793 METHODS FOR IN VITRO RECOMBINATION; Stemmer et al. U.S. Pat. No. 5,830,721 DNA MUTAGENESIS BY RANDOM FRAGMENTATION AND REASSEMBLY; Stemmer et al., U.S. Pat. No. No.
The inventors have developed applications of DNA shuffling technologies. Minshull et al. U.S. Pat. No. No. Crameri et al. (1996), ?Construction And Evolution Of Antibody-Phage Libraries By DNA Shuffling? Nature Medicine 2(1), 100-103, describes, for example, antibody shuffling in antibody phage library. Further details on DNA shuffling are also available in WO95/22625 WO97/20078, W098/3183, WO98/27230 WO97/35966 WO98/27230 WO98/13487 WO98/13485 WO989/42832.
Physical Nucleic Acid Shuffling Techniques” (as opposed to, e.g.?in silico’? Methods that are at least partially performed by manipulating character strings on a computer rely on actual recombination of physical nucleic acid, regardless of whether it is in vitro format or in vivo. Recombination is a common occurrence, for example, when complementary nucleic acid is present between the strands that are to be recombined. Thus. nucleic acids to be recombined are typically e.g., about 70% identical/complementary in sequence over regions of, e.g., about 30-40 nucleotides. It would be useful to be able recombine sequences with low or non-homologous homology to increase the sequence space encoded in recombinant RNAs resulting from shuffling. For example, for proteins which are commercially valuable, it would be desirable to be able to gain access to a recombination/mutation spectrum which is different than that of the native protein to provide for greater diversity in products produced by the various available shuffling strategies.
Likewise, nucleic acids recombination can be difficult modulate. This results in regions with high or low crossover frequencies between two different targets. The crossover frequency of a specific pairing of sequences between two different targets can be a feature that influences the recombinant products that are produced by recombination. To weight/bias the outcome of recombination products, it would be desirable to improve methods for modulating recombination frequencies at potential recombination site.
In general, it is highly desirable to develop new techniques that improve, enhance or add control levels to the recombination method. Techniques that allow shuffling divergent nucleic acid or modulation of the shuffling rate are particularly desirable. This invention also provides other features that will become apparent after reading this disclosure.
The present invention presents a number new formats of nucleic acids recombination for shuffling nucleic acids. The methods insert a number insertion sequences into one or multiple parental nucleic acids to produce a modified substrate nucleic for recombination. These insertions sequences can be shuffled by varying the number, type, and placement. These insertion sequences also allow for modulation or tuning of recombination frequencies between target nucleic acids. These methods typically use self-splicing, trans-splicing or cellular machinery to remove the insertion sequences from final coded nucleic acid or proteins. These methods use self-splicing and trans-splicing to remove insertions from coded nucleic acid or protein, for example, when the insertions are introns. These insertion sequences may also be markers, tags or other molecular elements, such as those used to purify encoded molecules, or allow the expression of toxic proteins. In a form that excises an intein in vivo, for example. In vitro expression can also result in the production in vitro of these proteins and others, for example, by using in vitro-expression systems.
The invention provides “methods of shuffling nucleic acids” (i.e. a first target and a secondary target nucleic acid). The methods provide a first target nucleic acids and a secondary target nucleic acids, e.g. by cloning or PCR amplification. Other methods are also possible. The first and second targets nucleic acids have at least one homologous or a non-homologous sequence of insertion nucleic acids, such as an intron, intein or subsequences removed by site-specific recombination, (e.g. similar to V-DJ recombination used for antibody production), etc. The target nucleics are recombined to produce a shuffled nucleic acid.
In addition, to new recombination techniques per se, the invention provides methods for producing selected proteins or RNAs for any purpose that these proteins and/or RNAs would normally be produced for. In one aspect, for example, a shuffled first nucleic sequence encoding the first portion of a selected protein, and a shuffled second nucleic sequence encoding the second portion of a selected protein, is provided. The nucleic additions can occur on the same strand as in cis-mediated reaction or on different strands, such as in trans-mediated reactions. The first and second sequences are combined to form a first subsequence of protein and a second subsequence of protein, which is then spliced together to create the desired protein. As described here, it is common to splice more than two sequences. For example, 3, 4, 5 6, 7, 8, 9 or 10 sequences. The splicing can occur in viro, in vivo, or both. “Splicing can be controlled or spontaneous.
In RNA production, a shuffled subsequence of nucleic acids encoding the first portion of selected RNA and a nucleic sequence encoding the second portion of selected RNA are also provided. These subsequences may be found on the same molecule or different molecules, depending on whether trans or cis splicing was used. The first and second subsequences of nucleic acids, or their RNA copies, are spliced together to produce a selected RNA. This RNA can then encode either a useful RNA such as an antisense or sense molecule or ribozyme, or it can encode a specific protein. The intein/RNA shuffling/production method can be combined, i.e. the spliced molecules of RNA can encode intein/extein sequences that are then spliced to produce useful proteins.
In general, a parent nucleic acids can be divided into exons (or exteins) by incorporation of several introns (or inteins) into the sequence. The target nucleic acids resulting from insertion sequences in the parental nucleic acids can, for example, have 5, 10, 15, 30, 50, or 100?mini exons? or ?mini exteins? Separated by a number of insertions.
In shuffling reactions, the first and second targets nucleic acids can be derived optionally from a parent nucleic acid and a target nucleic acid that are sufficiently distinct in sequence to prevent them from hybridizing in solution. The first and the second target nucleics can be obtained by integrating a plurality insertion sequences in the first or second parental nucleic acid. When the first two parental nucleic acids have been aligned to maximize identity, the first parental nucleic acid can be less than 50% identical, less than, for example, 40% or 30% or less, less, than, for instance, 25%.
Insertion nucleic acids can modulate the recombination frequencies between the first target and second nucleic acids. By inserting an intron in a parental sequence the recombination rate of subsequences of nucleic acids on either side of the parent can be reduced. In the same way, homologous mini-introns placed within parental sequences provide sites for recombination in the resulting targets.
Insertion Sequences can modulate expression in a cell type or cells, for example, when the insertions sequences contain one or more enhancers or other regulatory sequences.” Similarly, insertions sequences can also include splicing-enhancer sequences to facilitate splicing (e.g. ISEs such as the chicken heart troponin T ISE).
The Examples Gene
The methods described herein can be used for both virtual or in silico recombination and physical recombination. Recombination, for example, of character strings that represent nucleic acid, using a computer. After complete or partial sequences recombination, nucleic acid targets or nucleic acid derived from target nucleic acid can be synthesized. These synthetic nucleics can be recombined or cloned and selected, or manipulated as with any other nucleic acid.
A number of techniques can produce target nucleic acid containing insertion sequences.” These methods include chemical synthesis and PCR concatemerization. They also include in silico character strings generation or formation. In one embodiment, for example, the insertion of a plurality insertion nucleic acids sequences into one of more of the second and first parental nucleic acids sequences can be performed by physically connecting a plurality subsequences from the first or the second parental nucleics acid sequences.
As stated, the addition to parental nucleic acid of insertion sequences can modulate or modify the recombination resulting from target nucleic acid. The addition of insertions sequences to target sequences can also alter their hybridization properties. By adding insertion sequences in an appropriate number and arrangement, it is possible to make even non-homologous nucleic acid parental sequences hybridize. A target nucleic acids derived from parental sequences can also be made that does not hybridize to the parental nucleic acids under certain conditions (e.g. stringent hybridization). As mentioned above, these insertion sequences are useful for tuning recombination between certain regions of a nucleic acids, e.g. where a specific region is targeted to have an increased or reduced recombination.
The target and parental nucleic acid can have drastically different hybridization properties due to the presence of insertion sequences in the target nucleic acid. By including the target sequences in the target nucleic acids, the hybridization of the target nucleic acids to their parents can be prevented. Alternatively, a target sequence or sequences can be made to hybridize with one or more of the parent nucleic acid sequences to control the recombination characteristics of resulting shuffling nucleic acid reactions. In one embodiment, first and secondary parental nucleic acid sequencing hybridizes under strict conditions. However, first and two target nucleic sequences do not. In another embodiment, first and secondary parental nucleic acid do not hybridize in stringent conditions. However, first and two target nucleic acids do. In another embodiment, in which the first and the second nucleic targets nucleic acids hybridize, the first nucleic target does not undergo a hybridization under strict conditions with the second parental nucleic, or the second nucleic targeted does not undergo a hybridization under strict conditions with the first parental nucleic, In another embodiment, either the first or the second parental nucleic nucleic acid hybridizes under stringent condition to a third nucleic acid, while the first and the second target nucleic acids do not hybridize to the third nucleic acy. The number and arrangement will reveal a variety of modifications to hybridization.
Recombinant Nucleic Acids generated by recombining sequences of nucleic acid containing insertion subsequences may be recombined, shuffled or amplified in vivo, in vitro or synthesized using any laboratory or naturally-mediated techniques. In one embodiment, for example, a shuffled nucleic acids made by recombining a target nucleic with a number of insertion sequences, with one or several additional nucleic acids, is recombined to a third nucleic. Any selection method can be used to select the secondary shuffled nucleic acids for desired traits or properties. In general, any recombinant DNA can be selected to have a desired property or trait.
Recombinant Nucleic Acids are optionally expressed in cells or in vitro to produce a nucleic or protein. In one embodiment, expressed proteins can include extein and intein sequences. In most cases, the intein sequence (sometimes referred to an “intervening” protein sequence?) is excised from expressed proteins. The intein is removed from the expressed protein sequences. In parallel, the ligation (exteins), flanking sequences form a mature “extein protein”? Optionally, the ligated sequences (exteins) can be active in one, more, or all of these in vitro reactions or systems. So, expressed proteins can either be cleaved by proteolysis and ligated in order to produce a protein that is active or to remove an amino acid from an expressed protein. This ligation can be performed in both trans-splicing and cis-splicing formats. The reactions can occur in vitro or vivo, depending on whether the inteins are trans- or cis-spliced. Trans splicing introns and interins is explained further. see, Patten et al. ?ENCRYPTION of TRAITS using SPLIT GENESEQUENCES and ENGINEERED GENETIC ELEMENTS’? U.S. Ser. No. No. 60/164,618 filed Nov. 10, 1998.
The presence insertion sequences may be used to modulate the recombination rate between nucleic acid regions. Insertion sequences can be used to increase the frequency of cross-over between two points in a nucleic acid. It is useful, for example, when low linkage rates are desired between regions of nucleics to be recombined, or if one wants to evolve separate functional domains of elements of the nucleic.
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