Orthopedic Prometric MCQs - Chapter 3 Part 9

A retroviral vector derived from the Moloney murine leukemia retrovirus is among the best-developed viral vectors. Other viral vectors include adenovirus, adeno-associated virus, and herpes simplex virus. Novel vector systems based on lentivirus, a type of retrovirus, which includes human immunodeficiency virus (HIV), are being developed.

Novel approaches to treating genetic diseases involve gene repair or replacement rather than gene supplementation. One such approach is based on homologous recombination (replacement of a defective gene by a normal gene).

Adenoviral vectors can cause inflammatory reaction due to immune activation, an event linked to the first death related to gene therapy. This occurred in September 1999 at the University of Pennsylvania in a clinical trial in which an 18-year-old patient received infusion of over a trillion adenoviral vectors directed to his liver, which triggered a systemic inflammatory response that became uncontrollable, leading to organ failure and death. Newer generation gutted or gutlessâ adenovirus vectors are nonimmunogenic.

Two basic strategies exist for gene delivery. Direct, or in vivo, gene therapy involves direct introduction of vectors into the body. Indirect, or ex vivo, gene therapy involves removal of target cells from the body, vector introduction by incubation of the cells in vitro, and reimplantation. The in vivo system is less invasive.

Gene therapy has been suggested as a means of delivering sustained therapeutic levels of anti-arthritic gene products to diseased joints. Local gene delivery to the synovial tissue is preferred for osteoarthritis and other conditions affecting a few joints. It is less suited to address the extra- articular components of systemic conditions, such as rheumatoid arthritis. The gene that has been studied in greatest detail encodes the human IL-1 receptor antagonist (IL-Ra).

Identification of LIM mineralization protein-1 (LMP-1), a novel intracellular protein, is a step forward in osteoinductive proteins. Unlike BMP, which is a secreted protein that binds to cell-surface receptor to initiate a response, LMP-1 is an intracellular signaling molecule. Boden transfected bone marrow cells from rats ex vivo with LMP-1 gene using a DNA plasmid vector and used them during posterior thoracic and lumbar spine fusion in rats.

Various cytokines and cytokine antagonists hold promise as new therapeutic agents for osteoporosis. Baltzer et al showed that intramedullary injection of Ad-IL-1Ra gene in a murine ovariectomy model strongly reduced the loss of bone mass. Using a similar model, Bolon et al studied the effect of adenovirus-mediated transfer of osteoprotegerin, which showed more bone volume with reduced osteoclast numbers in axial and appendicular bones after 4 weeks compared to sham-operated mice.

In vivo gene delivery involves the direct injection of vectors containing the genes into the body with the expectation that they will reach and transduce the target cell.

Intervertebral disk degeneration has been associated with a progressive decrease in proteoglycan content of nucleus pulposus. The potential application of gene therapy for prevention of disk degeneration is to increase or maintain the proteoglycan content of nucleus pulposus. Thompson et al reported that addition of TGF-b1 to canine disk tissue in culture stimulated in vitro proteoglycan synthesis.

Neurofibromatosis is an autosomal dominant condition that occurs in a peripheral and central form. The peripheral type is more common and called neurofibromatosis 1 (NF1). The central type is characterized by bilateral acoustic neurofibromatosis (NF2). The clinical presentation is variable. Patients may either have a severe form of NF1 with hundreds of neurofibromas in the skin or they may have only cafa-au lait spots and deep neurofibromas that are easily apparent. Demographics of NF1 include: 1. Autosomal dominant with high degree of penetrance 2. C hromosomal abnormality - 17, NF1 gene (encodes neurofibromin) 3. Diagnostic criteria - two or more of the following: Six or more cafu au lait spots Two or more neurofibromas of any type or one plexiform neurofibroma Freckling in the axillary or inguinal area Optic glioma Two or more Lisch nodules Bone lesion First-degree relative with NF1

Neurofibromatosis is an autosomal dominant condition that occurs in a peripheral and central form. The peripheral type is more common and called neurofibromatosis 1 (NF1). The central type is characterized by bilateral acoustic neurofibromatosis (NF2). The clinical presentation is variable. Patients may either have a severe form of NF1 with hundreds of neurofibromas in the skin or they may have only cafa au lait spots and deep neurofibromas that are easily apparent. Demographics of NF1 include: 1. Autosomal dominant with high degree of penetrance 2. C hromosomal abnormality - 17, NF1 gene (encodes neurofibromin) 3. Diagnostic criteria - two or more of the following: Six or more cafa au lait spots Two or more neurofibromas of any type or one plexiform neurofibroma Freckling in the axillary or inguinal area Optic glioma Two or more Lisch nodules Bone lesion First-degree relative with NF1

In 1945, Grice developed the use of subtalar extra-articular arthrodesis as a treatment for paralytic valgus feet. Grice demonstrated that blocking the tarsal sinus opening prevents the calcaneus from rotating posterolaterally and thus blocks the heel of a foot from shifting into the valgus position. A bone block must be placed at such an angle in the tarsal sinus that when posterolateral migration of the calcaneus is attempted, the graft compresses. If a graft is placed too far forward, then the posterolateral movement of the calcaneus increases insertional distance between blocked areas, the graft collapses, and the foot goes into valgus.

The hamstrings are innervated at a level just proximal to the anterior tibialis muscle (with the highest innervated ankle motor at L5). Therefore, the patientâ s motor level is best estimated at L4.

Type 1 Monteggis fracture is associated with an anterior dislocation of the radial head and an apex anterior fracture of the ulna. Type 1 Monteggi s fracture is best treated by flexion and supination. The same maneuvers that reduce the ulna will also stabilize the radial head.

Although distal radial physeal fractures are common, growth disturbance is rare with a 1% occurence. Growth disturbance poses a challenge for follow- up, but physicians should at least mention the risk to patients.

Remodeling potential is significantly decreased at skeletal maturity. The first four scenarios represent skeletally immature patients. The 15-year-old girl should be treated like an adult to achieve the most acceptable result. All other patients deserve an attempt at closed reduction.

A tethered cord is common in patients with myelomeningocele. A tethered cord occurs due to adhesion of the dysplastic distal cord to its surrounding structures. The onset of a tethered cord may be exacerbated by closure of the open cord. Signs and symptoms of a tethered cord may include back pain, leg pain, increasing motor weakness, and increasing contracture. Alternate explanations for some of these signs and symptoms exist as well. An adult with Meryon's sign may drop a child he or she is holding due to weakness of the pectorals and latissimus muscles. Meryon's sign is also common in patients with myelomeningocele who have a C hiari's malformation. In this case, the patient with myelomeningocele does not experience weakness of the pectorals and latissimus muscles due to a tethered cord. Meryon's sign also occurs in some myopathies.

Patients with OI have a significant risk of basilar invagination. In patients with OI the skull settles around the foramen magnum and cervical spine, causing the brainstem to become indented. Signs and symptoms include increased spasticity (described here as muscle tightness), contractures, hyperreflexia, clonus, respiratory depression, and changes in voice or swallowing. Basilar invagination is most common in type IV OI. Although osteoarthritis is more common in patients with OI than in the general population at a given age, the 15-year-old patient in this case is too young to develop osteoarthritis. Spondylolysis would not produce this constellation of symptoms unless associated with severe listhesis.

As bowing becomes greater, the likelihood of a re-fracture increases. The presence of a lucent sclerotic line and pain increases the risk of re-fracture. Intramedullary fixation is preferred to plate fixation because plate fixation increases the risk of stress risers. Expandable rods that bridge the entire bone from one epiphysis to the other are the best treatment for excessive bowing. Examples include the Bailey Dubow or the Fassier rods.

Penetrating injuries to the sole of the foot commonly inoculate bacteria to the deep spaces of the foot. Pseudomonas, other gramnegative organisms, and Staphylococcus are examples of bacteria that commonly can become inoculated. An MRI would help to localize the site and stage of the infection. The MRI determines whether the surgeon should aspirate the infection and treat it with antibiotics or operatively debride the infection. Pseudomonas often requires debridement.

Lentiviruses are a subclass of retroviruses unique in their ability to transduce both dividing and non-dividing cells. This is highly advantageous for targeting mature, slow-dividing orthopedic tissues like articular cartilage.

Naked plasmid DNA delivery has the advantages of safety and low immunogenicity but suffers from very low transfection efficiency and transient gene expression. It typically requires physical methods like electroporation to improve cellular uptake.

AAV is a dependent parvovirus that requires a helper virus (like adenovirus or herpesvirus) to replicate. It is non-pathogenic, elicits minimal immune response, and typically remains episomal rather than integrating.

A promoter is a regulatory DNA sequence located upstream of a gene that binds RNA polymerase and transcription factors to initiate transcription. Tissue-specific promoters can restrict gene expression to target cells, such as osteoblasts.

Bone morphogenetic proteins (BMPs) bind to serine/threonine kinase receptors, leading to the phosphorylation of receptor-regulated Smads (Smad 1, 5, and 8). These complex with Smad 4 to enter the nucleus and regulate transcription of osteogenic genes.

siRNA mediates post-transcriptional gene silencing by binding to complementary messenger RNA (mRNA) sequences. This leads to mRNA degradation, effectively preventing translation into the disease-causing protein.

Ex vivo gene therapy involves harvesting cells, transducing them in vitro, and implanting them back into the patient. This allows for the selection and expansion of successfully genetically modified cells, ensuring high therapeutic efficacy.

In the CRISPR system, Cas9 acts as an endonuclease that creates site-specific double-strand breaks in DNA, guided by a specific RNA sequence. The cell's repair mechanisms are then exploited to correct the mutation.

OI Type I is typically caused by a null allele mutation resulting in haploinsufficiency, meaning a decreased amount of structurally normal collagen is produced. Missense mutations usually cause more severe types via dominant-negative effects.

Retroviruses utilize the enzyme reverse transcriptase to synthesize a complementary DNA (cDNA) strand from their RNA genome. This DNA is then integrated into the host genome via an integrase enzyme.

Adenoviral vectors deliver their genetic payload to the nucleus where it remains episomal (non-integrating), resulting in high-level but transient gene expression. They are also highly immunogenic.

FGFR3 is a negative regulator of bone growth. In a normal growth plate, its activation inhibits chondrocyte proliferation and hypertrophy. In achondroplasia, a gain-of-function mutation causes constitutive activation.

DMD is an X-linked recessive disorder. A male passes his Y chromosome to his sons and his X chromosome to his daughters. Therefore, an affected male will pass the mutation to all daughters (carriers) but no sons (0%).

A standard PCR cycle consists of denaturation (heating to separate strands), annealing (cooling to allow primers to bind), and extension (optimal temperature for DNA polymerase to synthesize the new strand).

Western blotting uses gel electrophoresis to separate proteins by size, followed by transfer to a membrane and probing with specific antibodies. Southern blots detect DNA, and Northern blots detect RNA.

DNA microarrays allow for the simultaneous measurement of the expression levels of thousands of genes (transcriptomics). This provides a global view of gene expression changes in tissues like a fracture callus.

Caspases (cysteine-aspartic proteases) are the central executioner enzymes in apoptosis. Their activation leads to the organized cleavage of cellular proteins, resulting in programmed cell death without eliciting an inflammatory response.

Antibiotic resistance genes in plasmids act as selectable markers. When the cell population is exposed to the antibiotic, only the cells that successfully took up the plasmid survive, allowing for their isolation.

Retroviruses integrate semi-randomly into the host genome. In early trials, this integration occurred near proto-oncogenes (insertional mutagenesis), inadvertently activating them and leading to clonal expansion and leukemia.

Electroporation uses brief, high-voltage electrical pulses to create transient pores in the cell membrane, allowing naked DNA to enter the cell. It is an effective non-viral, physical method of gene transfer.

Retroviruses integrate into the host genome, providing stable long-term expression but with a risk of insertional mutagenesis. Adenoviruses remain episomal.

Ex vivo gene therapy involves harvesting cells, transducing them in vitro, and implanting them back. This reduces the systemic immune response to the viral vector compared to direct in vivo injection.

AAV is a parvovirus that requires a helper virus (like adenovirus) to replicate. It has low immunogenicity and a relatively small packaging capacity (~4.7 kb).

Lipofection uses liposomes (lipid vesicles) to encapsulate DNA, allowing it to merge with the cell membrane and deliver genetic material. This method typically has very low immunogenicity compared to viral vectors.

Western blotting is used to detect and quantify specific proteins in a sample using antibodies. Southern blotting detects DNA, while Northern blotting detects RNA.

siRNA binds to specific messenger RNA (mRNA) molecules, leading to their degradation. This effectively silences the expression of the target gene post-transcriptionally.

Plasmid (non-viral) vectors are safer and less immunogenic than viral vectors but suffer from significantly lower transfection efficiency. Their gene expression is also typically transient.

IL-1 is a potent catabolic cytokine in osteoarthritis that upregulates degrading enzymes. Overexpressing IL-1Ra competitively inhibits IL-1, reducing cartilage degradation and MMP production.

Adenoviruses do not integrate into the genome, thus remaining episomal, and provide high but transient gene expression. However, they are highly immunogenic, which limits their repeated use in vivo.

Gel electrophoresis uses an electrical field to push negatively charged DNA molecules through a gel matrix. Smaller DNA fragments travel further and faster than larger ones, separating them by size.

Unlike standard retroviruses which require cells to be actively dividing to enter the nucleus, lentiviruses can transport their genome into the nucleus of non-dividing cells. This broadens their applicability in tissues like cartilage.

The guide RNA (gRNA) is designed to be complementary to the specific target DNA sequence. It guides the Cas9 enzyme to the correct locus to create a double-strand break.

Muscle-derived stem cells transduced with BMP-2 typically undergo a cartilage intermediate phase followed by osteogenesis. This process mimics endochondral ossification to form ectopic bone.

PCR cycles begin with denaturation of double-stranded DNA at high temperature (~94-98°C). This is followed by annealing of primers at a lower temperature, and finally extension by Taq polymerase.

Plasmids are extrachromosomal, circular, double-stranded DNA molecules found naturally in bacteria. They can replicate independently and are easily engineered to carry therapeutic genes of interest.

Mesenchymal stem cells (MSCs) are ideal vehicles for ex vivo gene therapy in orthopedics. They are easily harvested from bone marrow, expandable in culture, and possess osteogenic potential.

Vascular Endothelial Growth Factor (VEGF) is a primary driver of angiogenesis. This is a critical step in the early phases of fracture healing and endochondral ossification.

The bystander effect occurs when the therapeutic product (e.g., a toxic metabolite activated by a suicide gene) is transferred from transduced cells to neighboring untransduced tumor cells. This amplifies the tumoricidal effect.

DNA microarrays allow researchers to analyze the global gene expression profile by measuring thousands of mRNA transcripts simultaneously. They are often used to compare normal versus diseased tissue, such as OA cartilage.

Early human trials utilizing retroviral vectors for severe combined immunodeficiency (X-SCID) resulted in insertional mutagenesis near oncogenes like LMO2. This led to the development of leukemia in some patients, highlighting integration risks.

Retroviruses integrate their DNA into the host genome, allowing for stable, long-term expression, but they require the target cells to be actively dividing to gain access to the nucleus. Lentiviruses, a subclass of retroviruses, can infect both dividing and non-dividing cells.

AAV vectors provoke a very mild immune response compared to standard adenoviruses, allowing for more sustained gene expression in vivo. However, they have a limited packaging capacity (about 4.5 kb) and typically require a helper virus for replication.

The CRISPR-Cas9 system uses a synthetic guide RNA (gRNA) that is complementary to the target DNA sequence. This gRNA directs the Cas9 nuclease to the specific genomic location to induce a double-strand break.

Southern blotting is used to detect specific DNA sequences. The mnemonic SNoW DRoP helps differentiate the techniques: Southern=DNA, Northern=RNA, Western=Protein.

RT-qPCR is the most sensitive and quantitative method for measuring specific mRNA expression levels in cells. It first converts RNA to complementary DNA (cDNA) using reverse transcriptase, then amplifies and quantifies it in real-time.

Achondroplasia results from a gain-of-function mutation in the FGFR3 gene. This constitutively activates the receptor, which normally functions to inhibit chondrocyte proliferation, thus prematurely arresting endochondral ossification.

Runx2 (also known as Cbfa1) is the master transcription factor required for osteoblast differentiation. Mice lacking Runx2 completely lack bone due to a maturation arrest of osteoblasts. Sox9 is the equivalent master regulator for chondrocytes.

Ex vivo gene therapy involves isolating cells from the patient, modifying them genetically in a laboratory, and then implanting the transduced cells back into the patient. This allows for selection and quality control of the modified cells prior to implantation.

Sclerostin is a secreted glycoprotein that antagonizes Wnt signaling by binding to the LRP5/6 coreceptor on osteoblasts. This prevents the Wnt ligand from binding, leading to the degradation of β-catenin and a decrease in bone formation.

siRNA mediates RNA interference (RNAi), a process that leads to specific post-transcriptional gene silencing. The siRNA binds to a complementary target mRNA sequence, leading to its cleavage and degradation by the RNA-induced silencing complex (RISC).

Lentiviruses are a subclass of retroviruses with an RNA genome that reverse transcribes into DNA and integrates into the host genome. Unlike simple retroviruses, lentiviruses have the machinery to actively transport their pre-integration complex through the nuclear membrane of intact, non-dividing cells.

Fibrillin-1 normally binds to and sequesters latent TGF-β in the extracellular matrix. Mutations in FBN1 lead to failed sequestration, resulting in excessive TGF-β signaling, which drives many of the phenotypic features of Marfan syndrome.

The SLC26A2 gene encodes a sulfate transporter. Mutations cause defective intracellular transport of sulfate, leading to the undersulfation of proteoglycans in the cartilage matrix, which severely impairs cartilage structural integrity.

The high temperature (94-98°C) is used for the denaturation step in PCR. This breaks the hydrogen bonds holding the complementary DNA strands together, yielding single-stranded DNA required for primer binding in the subsequent annealing phase.

Western blotting is the standard technique for detecting specific proteins in a sample. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and incubating with specific antibodies.

Non-viral plasmid vectors are typically safer than viral vectors because they do not elicit a strong immune response and do not integrate into the host genome. However, their major limitation is low transfection efficiency and purely transient gene expression.

In dominant negative mutations, the defective gene product structurally interferes with the function of the normal protein from the wild-type allele. A missense mutation substituting glycine prevents proper folding of the entire type I collagen triple helix, leading to severe qualitative defects (Type II, III, IV OI).

DNA methylation, particularly at CpG islands within a gene's promoter region, generally leads to transcriptional repression or silencing. In contrast, histone acetylation is typically associated with open chromatin and active gene transcription.

Sox9 is the critical master transcription factor that drives the differentiation of mesenchymal stem cells into chondrocytes. Without Sox9, mesenchymal condensation and cartilage formation during endochondral ossification cannot occur.

Adenoviral vectors do not integrate into the host genome (reducing oncogenic risk) and can infect non-dividing cells. Their main clinical limitation is their high immunogenicity, which can elicit severe, sometimes fatal, inflammatory responses.

Southern blotting is used to detect specific DNA sequences in DNA samples. Northern blotting is used for RNA, and Western blotting is used to detect specific proteins.

Northern blotting detects specific RNA sequences (specifically mRNA) to study gene expression. It involves separating RNA fragments by size via electrophoresis and probing with a complementary sequence.

Western blotting is an analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. It relies on the use of specific antibodies to identify the target protein.

Standard retroviruses require target cells to be actively dividing for genome integration to occur. In contrast, lentiviruses (a subclass of retroviruses) can integrate into the genomes of non-dividing cells.

AAVs are highly favored for in vivo gene therapy because they elicit a very low immune response and cause no known human diseases. Adenoviruses, conversely, can provoke severe and highly destructive cellular immune responses.

Directly injecting a plasmid (which lacks viral components) into a living subject is a non-viral, in vivo method of gene delivery. While simpler and safer, it often suffers from lower transfection efficiency compared to viral vectors.

The Synthesis (S) phase of the cell cycle is when DNA replication takes place, resulting in the duplication of the cell's genetic content prior to entering the G2 phase and eventually Mitosis (M phase).

Ex vivo gene therapy involves isolating cells from a patient, cultivating and genetically modifying them in a laboratory setting, and then transplanting these optimized cells back into the patient.

The high-temperature denaturation step separates the double-stranded DNA into single strands. This exposes the target sequences, allowing primers to bind during the subsequent annealing phase.

siRNA utilizes the RNA interference (RNAi) pathway to achieve post-transcriptional gene silencing. It binds to complementary mRNA transcripts, leading to their degradation and preventing protein translation.

Achondroplasia is caused by a gain-of-function mutation in FGFR3, leading to its constitutive activation. This abnormal continuous signaling powerfully inhibits chondrocyte proliferation in the growth plate.

Reverse transcriptase is an RNA-dependent DNA polymerase. It is responsible for creating a complementary DNA (cDNA) strand from an RNA template, a critical step in RT-PCR and retroviral replication.

Severe forms of OI (Types II, III, IV) are usually caused by missense mutations (often substituting glycine) that produce abnormal collagen chains. These mutant chains disrupt the entire triple helix, exhibiting a dominant negative effect.

Adenoviruses do not integrate into the host genome but instead persist as episomes within the nucleus. This results in robust but transient expression of the transgene, alongside a strong host immune response.

Restriction endonucleases act as molecular scissors, cutting double-stranded DNA at specific recognition sites. The resulting fragments can then be ligated into vectors during cloning.

Unlike standard retroviruses, which require host cell division (nuclear envelope breakdown) to access the genome, lentiviruses possess pre-integration complexes that can cross the intact nuclear membrane of non-dividing cells.

The CRISPR system relies on a guide RNA (gRNA) specifically designed to be complementary to the target DNA sequence. The gRNA binds to the Cas9 enzyme and guides it to the precise genomic location to create a double-strand break.

Electroporation uses an electrical field to increase the permeability of the cell membrane, introducing pores through which plasmids or other macromolecules can pass. It is highly efficient for in vitro cell transfection.