Historical perspective

George Mendel first described the nature of inheritance in 1865 and thereby established the scientific field of genetics and opened the path for genetic discovery (Fig. 11.1). Mendel studied the inheritance pattern in pea plants and found that certain traits show up in offspring without any blending of parent characteristics.¹ For instance, the colors of the cross-pollinated plants, either white or purple, were not blended into intermediates in the offspring. However his work did not receive much attention until republished by others, notably Hugo de Vries, Carl Correns, and later by Bateson, who republished Mendel’s work in 1901. Bateson was the first to use the term “genetics,” and was also the main contributor to the rediscovery of Mendel’s research. Several conclusions could be drawn from the works of Mendel and Bateson to form the principles of mendelian or unifactorial inheritance. Genes, or factors according to Mendel’s terminology, come in pairs, one from each parent. Furthermore, genes can occupy different alleles, some of which are dominant while others are recessive (the principle of dominance). The principle of segregation, or Mendel’s first law, stated that alleles segregate from each other, one into each gamete, in the process of meiosis. The principle of independent assortment (Mendel’s second law) states that the segregation of different allelic pairs is independent.

Fig. 11.1 History. Timeline with the main milestones of genetic discovery.

The establishment of the principles of Mendel was followed by the search for the molecules responsible for the mechanisms of inheritance. Flemming first visualized the human chromosome in 1882. Thomas Hunt Morgan become the first to suggest that genes are located on chromosomes and his student, Sturtevant, used genetic linkage to demonstrate that genes are linearly arranged in chromosomes.^2,3

In 1924 Painter described the human diploid karyotype with 46 chromosomes, as well as the mechanism of XY sex chromosomes.⁴ Barr and Bertrand described the X-chromatin, or Barr body, in 1949. The fluorescent F body, or Y chromosome, was discovered 20 years after the publication of Barr’s paper.^5,6

Miescher took the first steps towards the discovery of the DNA molecule in the late 19th century. He was able to isolate phosphate-rich chemicals that he called nucleins from white blood cells. Another important contribution was that of Griffith, who discovered that dead bacteria could transfer genetic material to transform other bacteria. The transforming factor was later discovered to be DNA by Avery et al.⁷

Watson and Crick discovered the chemical structure of the DNA molecule in 1953.⁸ They used X-ray crystallography to determine the ladder-like double-stranded and coiled helix structure, composed of a phosphate deoxyribose sugar backbone and inward-pointing nucleotide bases. Their discovery was awarded the Nobel Prize in Medicine in 1962. The understanding of the structure of the molecule that carries genetic information laid the foundation for the development of new methodologies to investigate gene structure and function. In 1977 Sanger et al. invented DNA sequencing, a method to read the nucleotide sequence of DNA.⁹ In 1983 the group of Kary Banks Mullis described polymerase chain reaction (PCR), which enabled the isolation and amplification of specific stretches of DNA.¹⁰ These methodologies formed the basis for the sequencing of the entire human genome within the framework of the Human Genome Project.

Basic molecular biology

DNA

Genes are made of the molecule deoxyribonucleic acid, or DNA (Fig. 11.2). The DNA molecule is a long polymer of units called deoxyribonucleotides, or nucleotides. Each nucleotide is in turn composed of the phosporylated sugar phospodeoxyribose, and one of the four nucleotide bases adenine (A), guanine (G), cytosine (C), and thymine (T). Two such polymers form a two-stranded double helix with the sugar phosphates as hydrophilic backbones on the outside, and the hydrophilic bases on the inside. The bases of one strand bind to the bases of the other, so that A pairs with T and G pairs with C. As a consequence, the DNA chain is complementary and the sequence of one strand generates the sequence of the other. At one end the DNA chain starts with a sugar phosphorylated at its 5′ carbon and at the other end the chain ends with a 3′ hydroxylated carbon. The two chains are in antiparallel orientation with the 3′ end of one chains binding to the 5′ end of the complementary chain.

Fig. 11.2 Structure of the DNA molecule: the ladder-like double helix with the hydrophilic, phosphorylated sugar backbone on the outside and the hydrophobic nucleotide bases on the inside (left). The two strands of the molecule are complementary so that A binds with T and G with C (right). A, adenine; T, thymine; G, guanine; C, cytosine; P, phosphate.

Upon cell division DNA is synthesized in the process of replication. The complementary matching of nucelotide bases creates the basis for this process. Each DNA strand thus constitutes the template for the synthesis of an identical daughter DNA molecule. In eukaryotes, replication starts at multiple sites along the DNA molecule. Several DNA-binding proteins act to unwind the DNA double helix and separate the two strands, thereby opening up the molecule for replication. Synthesis starts with the formation of a RNA primer by the enzyme primase. The primer is a short complementary stretch of RNA that primes DNA synthesis by the enzyme DNA polymerase III. This enzyme creates phosphodiester bonds between the 3’ hydroxyl group of one sugar residue and the 5’ phosphate group of another. The sequence of the DNA template dictates the addition of nucleotides to the newly synthesized molecule. DNA polymerase has a built-in proofreading mechanism that removes nucleotides that have been incorrectly inserted during synthesis. Furthermore, DNA excising and repairing enzymes work to detect and correct DNA that has been damaged by various factors, such as oxygen radicals, radiation, and certain chemicals.

Organization of genomic DNA

One DNA molecule makes up one chromosome and each human chromosome contains about 1.3 × 108 basepairs. Human germ cells are haploid, which means they have one set of 23 different chromosomes, and somatic cells are diploid and contain 23 pairs, or 46 chromosomes. The autosomes are the 22 pairs that are not either of the two different sex chromosomes, X or Y. The 46 DNA molecules that make up the genome must be packed into the confined space of the somatic cell nucleus. This is accomplished with the help of DNA-binding proteins, or histones, and the consequent formation of chromatin (Fig. 11.3). Two of each histones 2A, 2B, 3, and 4 form a protein complex called the nucleosome. The DNA molecule winds around the nucleosomes and each nucleosome is separated by a piece of linker DNA. In this unfolded state this structure is viewed as “beads on a string” in the electron microscope. The nucleosomes are brought together by another histone, H1, to form the 30-nm chromatin fiber. Nonhistone proteins further condense the chromatin in a process called supercoiling. The condensed chromatin visible under the light microscope is called heterochromatin and the less compacted chromatin is called euchromatin. The chromosomes are most condensed just before mitosis. At this stage the cell is about to divide, the DNA has been replicated, and each chromosome is therefore composed of two chromatids. Each chromosme has a long (q) and a short (p) arm separated from each other by the centromere.

Fig. 11.3 Organization of genomic DNA. (A) The unfolded structure of the DNA double helix makes up the linker DNA, with short stretches of DNA in between nucelosomes. (B) The DNA molecule wraps around the histone structures called nucleosomes to form a structure viewed as “beads on a string” in the electron microscope. (C) The unfolded chromatin is packed to form the 30-nm chromatin fiber. (D–F) There is increased condensation from euchromatin to heterochromatin by the process of supercoiling. The mitotic chromosome is the most condensed form of chromatin.

Gene expression

A gene is a portion of the DNA molecule that contains information for the synthesis of gene products, which may be proteins or RNA molecules. In the diploid organism there are two alleles or copies of each gene, occupying the same locus, one on each chromosome. When the alleles are identical the organism is homozygous for that particular gene. Conversely, heterozygosis refers to different alleles at a particular genetic locus. The DNA sequence of the gene contains coding regions, exons, and noncoding regions, or introns. The information kept in the DNA molecule is transferred to an RNA molecule in the process of transcription. There are three major classes of RNA, messenger (m)RNA, transfer (t)RNA and ribosomal (r)RNA, and they all have different functions in the synthesis of proteins. The DNA sequence of genes encoding for a protein is transcribed into mRNA, which in turn forms the template for protein synthesis. This flow of information from DNA to protein is called the central dogma.

The RNA molecule has a similar chemical structure to DNA, albeit with a few differences. The sugar backbone in RNA is composed of ribose instead of deoxyribose, and the nucleotide base thymine (T) is replaced by uracil (U). Moreover, the RNA chain is a single-stranded molecule as opposed to the double-stranded DNA. When RNA is synthesized the sequence of bases depends on the sequence of DNA being transcribed. The DNA bases T, A, G, and C specify the bases A, U, C, and G, respectively, in RNA. Thus, the newly synthesized RNA molecule is complementary to the DNA template. The DNA/RNA sequence of a gene is composed of triplets of nucleotides called codons. Each codon specifies one amino acid and the way these triplets are read to specify the amino acid sequence of a polypeptide is called the reading frame. Start and stop codons constitute the initiation and terminations sites respectively for polypeptide chain synthesis. The enzyme RNA polymerase that recognizes the beginning of the gene sequence and creates phosphodiester bonds between the individual nucleotides catalyzes the transcription process. The nucleotide of the DNA template at the beginning of transcription is designated +1 and transcription is said to proceed downstream from this point, towards the 5’ end of the DNA strand. However, the RNA molecule is normally used as a reference and transcription is said to proceed from 5’ to 3’. Upstream nucleotides, designated with a negative number, form the DNA sequence up to the beginning of transcription. The DNA sequence just upstream of the gene is called the promoter. This sequence binds the RNA polymerase and various transcription factors upon initiation of transcription.

At termination of transcription the RNA strand is separated from the DNA template. In eukaryotes this process involves polyadenylation in the 3’ end of the RNA molecule. The mechanism, which separates the RNA from the DNA at termination of transcription in prokaryotes, is either a formation of a G–C-rich hairpin loop (Rho-independent), or the binding of a peptide factor called Rho (Rho-dependent).

In eukaryotes, mRNA transcripts undergo several modifications. The 5′ end is methylated to form the 7-methyl guanosine cap, while the 3′ end is altered by the above-mentioned polyadenylation to form the poly-A tail. Both the capping and the addition of the poly-A tail increase the stability of mRNA molecules towards degradation. Before the mRNA molecule can be used as a template for protein synthesis it undergoes the process of splicing, in which the parts of the mRNA sequence that correspond to introns, or noncoding regions, are removed. In alternative splicing, exons are rearranged in different ways to create different splice variants, or isoforms, of the protein in question. This constitutes an important regulatory mechanism by which cells can synthesize different variants of a peptide at different time points. However, alternative splicing to produce aberrant proteins is also highly implicated in disease. In syndromic craniosynostosis, mutations in the gene coding for FGFR2 may produce splice sites that lead to the synthesis of aberrant receptor protein.¹¹

Gene regulation

The control of gene expression is an extraordinarily complex process. The entire genome, with all its genes, is present in every somatic cell. However, the phenotype and behavior of cells during development, tissue repair, and homeostasis are directed by a differential and highly selective pattern of gene expression. The moment-to-moment alterations in gene expression are mainly regulated at the level of gene transcription. Cells respond and adapt with changes in gene expression to a wide range of stimuli from the external microenvironment, such as soluble factors, cell–cell contacts, or cell–matrix contacts. The effect of these stimuli on gene expression is in turn mediated by intracellular signal transduction mechanisms. Upon extracellular stimulation, complex cascades of intracellular enzymatic reactions are activated which ultimately result in the activation of a specific set of transcription factors (Fig. 11.4). Transcription factors bind to the promoter sequence and together with the RNA polymerase they form the preinitiation complex. Other transcription factors bind to so-called enhancer sequences, located upstream or sometimes downstream of the gene. The transcription factors encoded by homeobox-containing genes are particularly important for the developmental process.¹² A homeobox is a DNA sequence of about 180 basepairs that encodes for a so-called homeodomain, an amino acid sequence that binds DNA. Complicated molecular pathways involving several different growth factors regulate the timely and coordinated expression of genes during embryogenesis. Two growth factor families with particular importance in this regard are the transforming growth factor-β (TGF-β) superfamily and the fibroblast growth factors (FGF).¹³ Bone morphogenic proteins (BMP) are members of the TGF-β superfamily and modulate transcription of homeobox-containg genes, such as MSX1 and MSX2. MSX1 and MSX2 have both been implicated in the formation of orofacial clefts.¹⁴ Mutations in FGFR genes that lead to an increase in receptor signaling are the principal mechanisms behind FGFR-related craniosynostosis syndromes.¹⁵ The role of these factors in craniofacial development is elaborated below.

Fig. 11.4 Fibroblast growth factor receptor (FGFR) signal transduction pathways. The FGFRs are composed of an extracellular and an intracellular part. The extracellular part has three immunoglobulin domains (IGI, IGII, IGIII). FGF ligands linked to heparin sulfate proteoglycan (HSPG) bind to the FGFR which activates the receptor and leads to receptor dimerization. The activated receptor docks fibroblast receptor substrate 2 alpha (FRS2α), which leads to FRS2α phosphorylation at tyrosine residues. The next step is the activation of Grb2 and Sos. The Grb2–Sos complex activates Ras, which in turn phosporylates and activates Raf. Raf activates ERK, which enters the nucleus to phosphorylate and activate Ets transcription factors.

FGFR activation also leads to activation of phospholipase C-γ (PLCγ). PLCγ hydrolyzes phosphatidyl-inositol-4,5-diphosphate (PIP₂) to inositol-1, 4, 5-triphosphate (IP₃) and diaglycerol (DAG). IP₃ causes Ca²⁺ release while DAG activates Raf through activation of protein kinase c-δ (PKC δ). This in turn leads to Ras-independent MEK-ERK activation.

Epigenetic mechanisms

Stable variations in gene expression may be governed by other mechanisms than by actual variations in the primary DNA sequence. A number of so-called epigenetic mechanisms may modify the expression of genes and cause alterations in cellular phenotype. These mechanisms are preserved through cell division and are believed to be important for the stable tissue and cell-type-specific gene expression in differentiated cells. Moreover, epigenetic mechanisms may be inherited to affect the expression of phenotype. The investigation of epigenetic mechanisms has become a rapidly growing field of research with important implications for developmental biology as well as various disease processes. The so-called epigenotype is mainly formed by the three-dimensional organization of chromatin.

The three-dimensional architecture of chromatin regulates gene expression.¹⁶ Unfolding of the chromatin structure exposes the DNA sequence to transcription. Protein-encoding genes are as a consequence expressed in an environment of euchromatin and, conversely, the highly condensed heterochromatin inhibits transcription. The physical three-dimensional arrangement of chromatin is in turn regulated by different biochemical epigenetic mechanisms that may be transferred through cell division. The histones may be modified by methylations or acetylations. The pattern of methylation and acetylation of the histones in turn directs the binding of other chromatin-binding proteins that regulate gene transcription. Another mechanism is methylation of CpG dinucleotides of the DNA molecule by enzymes called DNA methyl transferases. DNA methylation generally silences gene transcription and the pattern of DNA methylations acts in concert with histone modifications to determine chromatin structure and regulate transcription (Fig. 11.5).

Fig. 11.5 Epigenetic mechanisms of DNA organization and restriction of gene transcription. The unfolded chromatin exposes the DNA molecule to the enzymes of gene transcription (top). Gene transcription is restricted by covalent modifications of DNA and histones. Bases in the DNA molecule are methylated by DNA methyltransferases. Methylated DNA restricts transcription by preventing transcription factors from binding to the DNA. Histone deacetylases modify histone proteins so that they become positively charged, which leads to binding to the negatively charged DNA and more compacted chromatin (middle). DNA methylation promotes binding of chromatin remodeling proteins that interact with histones to fold the chromatin into a more condensed state (bottom).

The epigenetic mechanisms give rise to so-called epigenetic phenomena.¹⁷ X-inactivation is the phenomenon by which one of the X chromosomes in females is randomly inactivated. The inactivated X chromosome can be visualized as a highly condensed chromatin structure, the Barr body. By inactivating one of the two X chromosomes in the somatic cells of females, the transcription of genes on the X chromosome, and consequent protein synthesis, is kept at the same level in males and females. During embryonic life paternal and maternal X chromosomes are inactivated in a random fashion. Therefore in any tissue there will be an equal number of cells with the active X chromosomes from either parent. This explains why most X-linked diseases are not expressed in females. Nonrandom X inactivation is an epigenetic abnormality that may lead to the occurrence of X-linked disease in females.

Gene regulation may depend on the sex of the parent from which the gene was inherited. Genomic imprinting is an epigenetic phenomenon that promotes the expression of only one parental allele. This is primarily accomplished by differential DNA methylation of alleles in a pattern specific to the parent of origin. Thus, an imprinted inherited gene is not expressed, which results in a departure from the unifactorial, mendelian pattern of inheritance for the trait directed by that gene. Uniparental disomy is a nonmendelian pattern of inheritance where there are two copies of a genomic sequence from one parent, and none from the other. If uniparental disomy occurs for imprinted genes, it may result in loss of function and disease.

Mendelian patterns of inheritance

Phenotypic traits determined by a single genetic locus are inherited according to the laws of unifactorial or mendelian inheritance. The inheritance of these traits may be either autosomal or sex-linked.

Autosomal inheritance

Autosomal inheritance refers to the transmission of traits directed by genes on any of the 22 pairs of autosomes. Autosomal inheritance may be either dominant or recessive (Fig. 11.6). Dominant traits are expressed in heterozygous states, while recessive traits are only expressed in individuals homozygous for that particular genetic locus. Thus, in autosomal-dominant disease the risk for the offspring to an affected individual is 50%. In autosomal-recessive disease, two parents, both heterozygous for the disease-causing gene, will have a 25% risk of having an affected child. Importantly, autosomal-dominant disease may arise as a result of inheritance, or from de novo gene mutation. A and B blood group antigens are both autosomal-dominant, but may be simultaneously expressed, a phenomenon referred to as codominance. Phenotypes determined by inherited or de novo mutated genes may be expressed to a varying degree. Thus, the same disease, caused by mutations in the same gene, may present with anything from very mild to severe manifestations. The explanation for this variable expressivity may be caused by different mutations in the same gene, other modifying genetic and molecular mechanisms, or environmental factors. Nonpenetrance refers to the situation when individuals heterozygous for an autosomal-dominant trait do not express the corresponding phenotype. This explains why autosomal-dominant disease may skip generations to reappear in coming generations.

Fig. 11.6 (A) Autosomal-dominant pattern; (B) autosomal-recessive inheritence pattern.

(Reproduced with permission from Cohen MM. Dysmorphology, syndromology, and genetics. In: McCarthy JG (ed.) Plastic Surgery. Philadelphia: WB Saunders, 1990:105, 106.)

Sex-linked inheritance

The genes on the X and Y chromosomes are inherited according to a pattern of sex-linked inheritance. X-linked dominant traits may be expressed in both males and females, but will never be transmitted from affected father to son (Fig. 11.7). Daughters with affected fathers will on the other hand have a 100% risk of inheriting the trait. Affected mothers will have a 50% risk of transmitting an X-linked dominant trait to both daughters and sons. X-linked recessive traits are much more common and are almost exclusively expressed in males, as the two copies of the X chromosome in females, one from each parent, will usually ascertain heterozygocity. Examples of X-linked recessive disease are Duchenne’s muscular dystrophy, Becker’s muscular dystrophy, and hemophilias A and B. Y-linked inheritance affects only males, exemplified by inheritance of the H-Y histocompatibility antigen.

Fig. 11.7 (A) X-linked dominant pattern; (B) X-linked recessive pattern.

(Reproduced with permission from Cohen MM. Dysmorphology, syndromology, and genetics. In: McCarthy JG (ed.) Plastic Surgery. Philadelphia: WB Saunders, 1990:105, 106.)

Nonmendelian patterns of inheritance

Nonmendelian inheritance refers to specific patterns that do not follow the principles of mendelian inheritance. One example is the inheritance governed by the epigenetic phenomenon of imprinting described above. Other patterns are uniparental disomy and mitochondrial inheritance.

Mutations

Most interindividual differences in the genetic code do not cause disease. In fact, the DNA sequence is highly variable between any two individuals. This variability is created by so-called polymorphisms, variations in the genetic code that in themselves normally do not give rise to disease. However, certain patterns of polymorphisms may be related to increased risk of disease and variable response to environmental factors. The most common form of polymorphism is single nucleotide polymorphism, or SNP. SNPs are nucleotide substitutions that occur in 1 out of approximately 300 nucleotides in coding and noncoding DNA. The SNPs can be used as markers for specific regions of the genetic code and so-called SNP maps have been extremely useful in genetic studies and diagnosis.

The most common forms of genetic mutations are single nucleotide substitutions. Other examples are deletions or insertions of several nucleotides. Mutations may result in amino acid substitutions (missense mutation). The altered amino acid sequence may in turn cause structural or functional modifications of the protein. These modifications may cause disease by several different mechanisms. The physiologic activity of the protein may be abolished (loss of function) or increased (gain of function). Alternatively, structural modifications may alter target functions for other proteins or create dysfunctional polypeptide complexes. Gene mutations may alter gene expression at various levels. Mutations in gene promoters or other sequences that bind transcription factors may increase or decrease transcription, while mutations in introns may affect the splicing of mRNA transcripts. Other mutations may alter the stability of the mRNA molecule, leading to increased degradation or accumulation of transcripts. Mutations may also affect protein synthesis by interfering with initiation and termination of translation. The so-called nonsense mutations introduce a stop codon, which prematurely terminates protein synthesis and leads to a truncated protein product. Frameshift mutations alter the reading frame and may lead to the synthesis of abnormal polypeptide chains. Such malformed proteins are frequently susceptible to proteolytic degradation.

Inherited chromosomal abnormalities

Errors in disjunction of chromosomes during meiosis or mitosis may cause an abnormal number of chromosomes, or a numerical abnormality. The most common abnormality seen in human disease is aneuploidy, or a number of chromosomes that is not a multiple of the haploid set of chromosomes. Aneuploidy in humans is most often due to an abnormal number of sex chromosomes. Supernumerary X chromosomes may lead to increased dosage of gene products from the X chromosome. However, if all but one of the X chromosomes are inactivated, the effect on phenotype may be very slight. Monosomy for X chromosome, or 45X, is the most common of all numerical abnormalities. However, 45X usually leads to abortion in the first trimester. Autosomal numerical abnormalities are usually in the form of trisomy. The most common of all chromosomal conditions is Down syndrome, caused by trisomy 21. Certain chromosomal sites are prone to various rearrangements that produce structural chromosomal abnormalities. Segments of chromosomes may be deleted or duplicated. CATCH 22 (see below) is a syndrome that may include abnormalities of the palate and is caused by deletion of locus 22q11.¹⁹ Other types of structural abnormalities are translocations, where there is an exchange of segments between chromosomes, and inversions, referring to a situation when a chromosomal segment is detached, inverted, and reinserted.

Gene delivery

Gene delivery is achieved through physical, chemical, and biological approaches.²³ All these approaches can be used in vivo or ex vivo. Transgene delivery in the form of naked DNA is often less efficient mainly due to rapid degradation by nucleases. Therefore, various specialized technologies have been developed to increase transfection efficiency. The ideal gene delivery approach should: (1) allow for delivery of appropriate transgene size; (2) evade immunologic/toxic reactions or other detrimental effects; (3) allow for target cell specificity; (4) produce efficient delivery; and (5) provide gene modification of appropriate duration.

There are nonbiological and biological methods for gene delivery. The nonbiological methods may in turn be divided into needle injection of naked DNA, physical and chemical methods.²⁴ Physical approaches, such as microseeding, electroporation, ballistic, ultrasound or hydrodynamic delivery, employ a physical force to deliver the transgene into the cell through the plasma membrane. Chemical approaches use synthetic or naturally occurring chemical compounds as transgene carriers. The predominating type of nonbiological method is chemical, through delivery by cationic liposomes. The liposomes are synthetic vectors composed of positively charged cationic lipids and co-lipids, which may be noncharged phospholipids or cholesterol. These liposomes are mixed with transgene-containing plasmids to form particles called lipoplexes. The DNA bound in these particles is resistant against degradation by nucleases. Cellular uptake is presumed to occur through endocytosis and the DNA exits the intracellular vesicles before degradation in lysosomal compartments (Fig. 11.9).

Fig. 11.9 DNA delivery to cells using cationic liposomes. Cationic liposomes form complexes (lipoplexes) with negatively charged DNA through electrostatic interactions. The particles enter the cells through the process of endocytosis. The endosomal membrane is destabilized in the cytosol and the DNA molecule enters the nucleus.

Liposomes have been of limited value for in vivo gene therapy mainly due to a risk of toxic reactions and rapid plasma clearance. Other examples of chemical methods are receptor-mediated and cationic polymers. Viral gene delivery is the predominating form of biological gene delivery.²⁵ Commonly used viruses are adenoviruses, herpes simplex viruses, and retroviruses. Viral gene delivery is based on the natural ability of viruses to infect host cells with genetic material. In order to be used as vectors, viruses first have to be genetically modified. The genes required for viral replication are deleted and replaced by the gene of interest. To compensate for this inability to replicate, the viral vectors are propagated in packaging cell lines that provide the genes encoding for the structural proteins for the viral particle. In general, the viral approaches are associated with higher transfection efficiency and more stable transformations. The main concerns with biological methods are the risks of transmitting disease, inducing insertional mutations, or evoking immunological or toxic reactions. In retroviral vectors, the retroviral single-stranded RNA genome is reverse-transcribed into double-stranded DNA and the transgene is integrated into the genomic DNA of host cells. This offers the possibility of stable transfection, however, with a risk for insertional mutations (Fig. 11.10). The risks of toxicity of viral gene therapy were clearly demonstrated by the tragic death of an 18-year-old male patient treated for ornithine transcarbamylase deficiency.²⁶

Fig. 11.10 Retroviral gene transfer. The retroviral single-stranded RNA genome is reverse-transcribed into double-stranded DNA and the transgene is integrated into the genomic DNA of host cells. Retroviruses offer the possibility of stable transfections.

Immunosuppression has been used to reduce immunological responses to viral vectors. However, this strategy considerably increases the risks and side-effects of the treatment. Nonviral biological vectors such as bacterial, bacteriophages and virus-like particles may become valuable delivery tools in certain gene therapy niches.²⁷ Today, about two-thirds of clinical gene therapy trials are based on viral vectors, and merely 5–10% deploy lipofection as a mode of gene delivery.²² Features of the various techniques for gene delivery are summarized in Table 11.1. There is a need for more efficient and safer methods for gene delivery, and ongoing research aims at the development of improved technologies for delivery.

Table 11.1 Common techniques for gene delivery

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

Technology innovation in plastic surgery Cold and chemical injury to the upper extremity Botulinum toxin (BoNT-A) Free TRAM breast reconstruction Facelift Facial transplant

Stay updated, free articles. Join our Telegram channel

Join

Tags: Plastic_Surgery_E-Book__6_-_Volume_Set__Expert_C

Feb 21, 2016 | Posted by admin in General Surgery | Comments Off on Genetics and prenatal diagnosis

Full access? Get Clinical Tree

Get Clinical Tree app for offline access

Get Clinical Tree app for offline access

Gene delivery technique	Advantages	Disadvantages
Direct injection of naked DNA/plasmid DNA	Simple, local delivery, unlimited gene size, nontoxic and nonimmunogenic	Only direct injection, very low transfection efficiency, transient gene expression only
Gene gun	Can deliver large amounts of DNA; technically simple	Nonspecific, physical damage to cell required for DNA uptake, low transfection efficiency, potential foreign-body reaction
Microseeding	Can deliver large amounts and different types of DNA	Low transfection efficiency, cellular damage, limited experience
Electroporation	Large transgene size, nontoxic	Nonspecific, complex equipment, damage to cell membrane required for DNA uptake
Cationic liposomes	Technically simple, can transfect any cell type, large transgene size, local delivery, low immunogenicity	No targeting, toxicicity, low transfection efficiency, transient transfection, rapid plasma clearance in vivo
Retrovirus	Tranduces many different cell types, high efficiency of ex vivo transduction, long-term gene expression	Transduces mainly dividing cells, inefficient transduction in vivo, risk of insertional mutagenesis, limited transgene size, difficult to propagate in culture
Adenovirus	Transfects virtually all cell types, dividing and nondividing cells, good transfection efficiency in vitro and in vivo, no integration into host genome, large transgene size	Immune response, lack of permanent expression, pre-existing antibodies are common, potential wild-type breakthrough infection
Adeno-associated virus	Transduces dividing and nondividing cells, integrates to specific site at chromosome 19, long-term gene expression, low immunogenicity	Difficult to grow to high titers, risk of insertional mutagenesis, limited transgene size, pre-existing antibodies, complex and expensive to manufacture
Hepres simplex virus 1	Transduces wide variety of cell types, neurotropism, large transgene size, long-term expression feasible, low immunogenicity